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The use of radioactive materials (RAM) requires prior approval by a designated safety committee at each institution.
1. Preparation of stock solutions for&#160;14C-labeled substrates
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	<li>Make 4 mM bovine serum albumin (BSA) stock solution by mixing 11 g of BSA and 33.1 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS) and shaking it gently at room temperature for ~3 h until BSA is fully dissolved. Warm the BSA stock solution in a 70 &#176;C water bath before use.</li>
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substrate oxidation assay in cultured cells: an in vitro assay to quantify substrate oxidation in cells by measuring radioactive signals from trapped co2

Source: Song, C. et al., <a href="https://www.jove.com/v/63568">Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping.</a> J. Vis. Exp. (2022)
This video demonstrates a quantification technique for CO2 released during substrate oxidation using 14C-radiolabeled substrates. The released 14CO2 is trapped in an alkaline solution and quantified by a scintillation counter. The oxidation of different substrates varies between tissues and reflects the pathophysiological condition of the tissue.

<img alt="Figure 1" src="/files/ftp_upload/21162/21162_Figure1.jpg" /> 
Figure 1: Diagrams for CO2 trapping assay and calvaria dissection. (A) The middle region of the calvaria, indicated by the orange dashed triangle, is harvested for cell isolation. (B) 1.5 mL microcentrifuge tubes are used for CO2 trapping (step 1). Filter papers (blue paper for illustration purposes) are cut to fit into tube caps (step 2). A...

Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO2

Source: Song, C. et al., Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping. J. Vis. Exp. (2022)
This video demonstrates a quantification ...

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.

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This video demonstrates a quantification technique for CO2 released during substrate oxidation using 14C-radiolabeled substrates. The released 14CO2 is trapped in an alkaline solution and quantified by a scintillation counter. The oxidation of different substrates varies between tissues and reflects the pathophysiological condition of the tissue.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21162/21162_Figure1.jpg" /> 
Figure 1: Diagrams for CO2 trapping assay and calvaria dissection. (A) The middle region of the calvaria, indicated by the orange dashed triangle, is harvested for cell isolation. (B) 1.5 mL microcentrifuge tubes are used for CO2 trapping (step 1). Filter papers (blue paper for illustration purposes) are cut to fit into tube caps (step 2). A...

Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO₂

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Overview

Protocol

Representative Results