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

Published: April 30, 2023

Abstract

Source: Song, C. et al., Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping. 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.

Protocol

The use of radioactive materials (RAM) requires prior approval by a designated safety committee at each institution. 1. Preparation of stock solutions for 14C-labeled substrates Make 4 mM bovine serum albumin (BSA) stock solution by mixing 11 g of BSA and 33.1 mL of Dulbecco'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 °C water bath before use. <…

Representative Results

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…

Declarações

The authors have nothing to disclose.

Materials

0.22 µm filters Sigma-Aldrich SLGVM33RS Used to filter BSA solution
0.25% Trypsin-EDTA Gibco 25200056 Dissociate cells from cell culture
plates
1.5 mL Eppendorf tubes PR1MA PR MCT17 RB Used for reaction incubation
10 cm plates TPP 93100 Used for cell culture
10 mL syringe BD 302995 Used to flush marrow from long
bones
10% FBS Atlanta biologicals S11550 For Cell culture medium preparation
14C -Glucose PerkinElmer NEC042X050UC Used to make hot media
14 C-glutamine PerkinElmer NEC451050UC Used to make hot media
14 C-oleate PerkinElmer NEC317050UC Used to make hot media
23 G needle BD 305120 Used to flush marrow from long
bones
24-well plates TPP 92024 Used for cell culture
70 μm cell strainers MIDSCI 70CELL Used to filter supernatant during
cavarial digestion
Acridine Orange/Propidium Iodide
(AO/PI) dye
Nexcelom Biosciences CS2-0106 Stains live cells to determine seed
density
Bovine Serum Ablumin Proliant Biologicals 68700 Used for fatty acid conjugation
Cellometer Auto 2000 Nexcelom Biosciences Determine the number of viable cells
Centrifuge Thermo Fisher Legend Micro 21R Used to pellet cells
Collagenase type II Worthington LS004176 Dissociate cells from tissue
Custom MEM alpha GIBCO SKU: ME 18459P1 Used to create custom hot media
Dulbecco's Phosphate-Buffered
Saline
Gibco 10010023 Used to dissolve and dilute reagents,
and wash culture dishes
Filter Paper Millipore-Sigma WHA1001090 Traps CO 2 with sodium hydroxide
Glucose Sigma-Aldrich g7528 Used to make custom media
HEPES Gibco 15630080 Traps CO 2 during cell culture
L-carnitine Sigma-Aldrich C0283 Supplemented for fatty acid oxidation
L-Glutamine Sigma-Aldrich g3126 Used to make custom media
MEM alpha Thermo A10490 Cell culture medium
Parafilm Pecheney Plastic Packaging PM998 Used to seal cell culture dishes
Penicillin-Streptomycin Thermo Fisher 15140122 Prevents contamination in cell culture
Perchloric Acid Sigma-Aldrich 244252 Releases CO 2 during metabolic
assay
Pyruvate Sigma-Aldrich p5280 Used to make custom media
Scintillation Counter Beckman Coulter LS6500 Determines radioactivity from the
filter paper
Scintillation Fluid MP Biomedicals 882453 Absorb the energy emitted by RAMs
and re-emit it as flashes of light
Scintillation Vial Fisher Scientific 03-337-1 Reaction containers for scintillation
fluid
Sodium carbonate Sigma-Aldrich S5761 Balance buffer for medium
Sodium Hydroxide Sigma-Aldrich 58045 Traps CO2 during metaboilc assay
Sodium oleate SANTA CRUZ SC-215879 BSA conjugated fatty acid
preparation
Vaccum filtration 1000 TPP 99950 Filter cMEMα

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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. J. Vis. Exp. (Pending Publication), e21162, doi: (2023).

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