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Adaptation of the technique of determination of mitochondrial membrane potential change in C2C12 cells using the ready set “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich)

https://doi.org/10.18705/2311-4495-2024-11-1-45-54

EDN: AXQBHV

Abstract

   Background. We used the “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich) to detected MMP but encountered difficulties by applied this kit because in manual there not JC-10 concentration and it not allowing for account cell type, size, density, differences in incubation time for different cell cultures.

   Objective. Adaptation of the method for determining MMP (mitochondrial membrane potential) in C2C12 cells using in microplate reader and electron microscope.

   Design and methods. MMP in C2C12 cells was measured by two way, using fluorescence microscopy (Zeiss, Zen program) and using a plate fluorimeter (CLARIOstar (BMG LABTECH). JC-10 and TMRE dyes (Sigma-Aldrich) were used as fluorescent probes.

   Results. Optimal conditions for detection changes in mitochondrial membrane potential in C2C12 cells were selected. 100- fold dilution of the dye JC-10 (Dye Loading Solution) and replacement of the manufactured buffer to PBS led to repeatability and reproducibility results.

   Conclusion. When using ready-made kits for measuring MMP, the method proposed by the manufacturer may not be suitable for the selected cell line. In our study to mouse myoblasts of the C2C12 line, a dilution of the dye for loading was required 100 times compared to that recommended by the manufacturer.

About the Authors

Yu. A. Vlasova
Almazov National Medical Research Centre
Russian Federation

Yulia A. Vlasova, Cand. Sc. (Biology), Senior researcher

Research Laboratory of Molecular and Cellular Modeling and Gene Therapy

197341; Akkuratova str., 2; Saint Petersburg



E. S. Klimenko
Almazov National Medical Research Centre
Russian Federation

Ekaterina S. Klimenko, Junior Researcher

Research Laboratory of Molecular and Cellular Modeling and Gene Therapy

Saint Petersburg



K. S. Sukhareva
Almazov National Medical Research Centre
Russian Federation

Ksenia S. Sukhareva, PhD, Junior Researcher

Research Laboratory of Molecular and Cellular Modeling and Gene Therapy

Saint Petersburg



L. S. Gavrilova
Almazov National Medical Research Centre
Russian Federation

Lidiya S. Gavrilova, Master’s student

Institute of Medical Education

Saint Petersburg



A. A. Kostareva
Almazov National Medical Research Centre
Russian Federation

Anna A. Kostareva, MD, PhD, Director of the Institute, Professor

Institute of Molecular Biology and Genetics; Institute of Medical Education; Department of Faculty Therapy with the Clinic

Saint Petersburg



References

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2. Perry SW, Norman JP, Barbieri J, et al. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques. 2011;50(2):98–115. DOI: 10.2144/000113610.

3. Mathur A, Hong Y, Kemp BK, et al. Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Cardiovasc Res. 2000;46(1):126–38. DOI: 10.1016/s0008-6363(00)00002-x.

4. Buckman JF, Reynolds IJ. Spontaneous changes in mitochondrial membrane potential in cultured neurons. J Neurosci. 2001;21(14):5054–65. DOI: 10.1523/JNEUROSCI.21-14-05054.2001.


Review

For citations:


Vlasova Yu.A., Klimenko E.S., Sukhareva K.S., Gavrilova L.S., Kostareva A.A. Adaptation of the technique of determination of mitochondrial membrane potential change in C2C12 cells using the ready set “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich). Translational Medicine. 2024;11(1):45-54. (In Russ.) https://doi.org/10.18705/2311-4495-2024-11-1-45-54. EDN: AXQBHV

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ISSN 2311-4495 (Print)
ISSN 2410-5155 (Online)