This article describes a method for measuring the rate of substrate oxidation in metabolically active cells using radiolabeled carbon-14. The process involves incubating cells with a radioactive substrate and quantifying the resulting carbon dioxide production.
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.
Wash the cells in the extra wells, twice with DPBS. Dissociate the cells with 0.25% trypsin-EDTA. Resuspend 20 microliters of digested cells in DPBS with 20 microliters of acridine orange and propidium iodide dye solution. Determine the number of live cells with an automated cell counter, and record the number.
Wash the cells in the assay wells twice with DPBS. Add 500 microliters of hot medium to each assay well in the RAM-designated tissue culture hood. After sealing the plate with parafilm, incubate the cells in an RAM-designated incubator at 37 degrees Celsius for four hours.
During incubation, cut filter paper into circular pieces slightly bigger than the area inside the cap of the 1.5-milliliter microcentrifuge tube, and insert the paper snugly into the cap. Add 200 microliters of 1 molar perchloric acid to each tube, and 20 microliters of sodium hydroxide to the filter paper fitted inside the cap.
After incubating the cells, transfer 400 microliters of culture medium from each well to the prepared tubes, and close the caps immediately. Leave the tubes in a tube rack at room temperature for 1 hour.
Parallelly, during incubation, set up a scintillation vial for each tube, and fill it with 4 milliliters of scintillation fluid. Transfer each piece of filter paper to a scintillation vial, and incubate at room temperature for 30 minutes.
Perform wipe tests on the tissue culture hood, the water bath, refrigerator, incubator, sink, ground, and any other working area for potential RAM contamination. Put the paper wipes into scintillation vials containing scintillation fluid.
Measure carbon-14 radioactivity in the scintillation vials with a scintillation counter, and record the reading results. Decontaminate the working environment according to radiation safety guidelines, if necessary.
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