Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO2
Transcription
In metabolically active cells, carbon-containing substrates are oxidized via a series of biochemical reactions to generate energy and release carbon dioxide as a by-product.
To determine the rate of substrate oxidation, begin with an adherent culture of the desired undifferentiated cells. Supplement the cells in a medium with a suitable substrate containing radioactive carbon fourteen in its backbone, and incubate.
The cells consume the substrate and oxidize it to release radiolabeled carbon dioxide, which diffuses out of the cells into the surrounding medium.
Transfer this medium into a gas-trapping assembly containing perchloric acid to quench the reaction. This tube contains a sodium hydroxide-soaked filter paper fitted inside the tube cap. Close the cap, and incubate.
During incubation, the carbon dioxide, in its gaseous form, releases inside the tube. Sodium hydroxide absorbs the radiolabeled carbon dioxide, entrapping it in the filter paper. Transfer this filter paper into a vial containing a scintillation cocktail, and position it inside the liquid scintillation counter.
Inside the counter, radiolabeled carbon undergoes radioactive decay, emitting high-energy beta particles. These particles get absorbed by the components of the scintillation cocktail and emit light pulses, which are computed by the scintillation counter.
The amount of radioactivity detected correlates with the rate of substrate oxidation in the cells.
