# In Vitro Antioxidant Effects of Coenzyme Q10 on Cellular Metabolism in Aged Mesenchymal Stem Cells
## 2 Materials and Methods
### Assessment of Fatty Acid Profile  
To evaluate the fatty acid composition following short-term treatment with CoQ10, 1 \(\times\) 10\({}^{6}\) cells per experimental condition were processed. The protocols of extraction used in this method were adapted after previous publications of the group [25; 27]. All solvents and reagents used in the extraction, derivatization, and analysis were GC-grade and obtained from Sigma-Aldrich (St. Louis, MO, USA).  
Cells were washed with ice-cold PBS to remove residual media and centrifuged at 1500 rpm for 10 min to form a pellet used further for lipid extraction. Lipid extraction was performed by resuspending the cell pellet in 400 \(\upmu\)L of a chloroform/methanol solution (2:1 \(v\)/\(v\)), and the mixture was centrifuged at 4000 rpm for 5 min. The lower (chloroform) layer was transferred to a clean tube, and 200 \(\upmu\)L of PBS and 100 \(\upmu\)L of chloroform were added, followed by vortexing and a second centrifugation at 4000 rpm for 10 min. The lower (chloroform) phase was collected, evaporated to dryness under a nitrogen stream, and stored for subsequent analysis. The dried residue was subjected to saponification by adding 0.45 M sodium hydroxide and heated at 65 \({}^{\circ}\)C for 1 h. The resulting solution was neutralized with 0.45 M hydrochloric acid, and 2% acetic acid in hexane was added for extraction two times. The upper (hexane) layers were pooled and dried under nitrogen gas. The resulted residue was derivatized with 100 \(\upmu\)L of acetyl chloride in 5 mL methanol under continuous agitation at room temperature for 45 min. The reaction was stopped by adding 3 mL of 0.25 M potassium carbonate. Then, 1 mL of hexane was used to extract the resulting fatty acid methyl esters (FAMEs) under continuous agitation at room temperature for 1 h. The resulted upper (hexane) layer was also dried under inert nitrogen gas and stored at -20 \({}^{\circ}\)C for further GC-MS analysis.  
The resulting residue containing FAMEs was resuspended in 100 \(\upmu\)L hexane and was analyzed using an Agilent 6890 gas chromatograph coupled with a 5973 MSD quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved using a DB-WAX capillary column (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m) with helium as the carrier gas at a flow rate of 1 mL/min. The oven temperature started from 50 \({}^{\circ}\)C to 250 \({}^{\circ}\)C, with a rate of 6 \({}^{\circ}\)C/min. Then, 1 \(\upmu\)L of each sample was injected into an inlet in splitless mode,the vaporization temperature being 230 \({}^{\circ}\)C and the mass spectrometer source being set at 150 \({}^{\circ}\)C.  
The mass spectra of the compounds were monitored in full-scan mode after a solvent delay of 4 min, and the mass range was set between 50 and 600 Da. The ionization energy was set to 70 eV. The identification of the compounds was made by comparing their spectra to the NIST11 mass spectral library using ChemStation software, version B.01.00 (Agilent Technologies, Palo Alto, CA, USA). Solvent blanks and control samples were analyzed under the same GC-MS conditions to exclude contamination and ensure data accuracy. The area percentage of each FAME was calculated as follows: the peak area for a FAME was divided by the summed areas of all the FAME peaks in the chromatogram.