|Zujovic Tijana, Krako Jakovljevic N, Pavlovic K, Markovic I, Lalic NM (2019) Palmitate treated human hepatocellular carcinoma HuH7 cells require higher digitonin concentration for plasma membrane permeabilization. MitoFit Preprint Arch doi:10.26124/mitofit:ea19.MiPSchool.0003.|
Abstract: Version 1 (v1) 2019-06-04 doi:10.26124/mitofit:ea19.MiPSchool.0003
Mitochondrial physiology analysis is central to cell metabolism research and understanding of many human diseases. High-resolution respirometry represents an efficient approach to study mitochondrial physiology in isolated mitochondria, both permeabilized and intact cells, as well as in tissue biopsy samples. We use digitonin for controlled plasma membrane permeabilization to perform extended functional OXPHOS analysis by O2k Oxygraph in palmitate treated cells, an in vitro model of insulin resistance and lipotoxicity. Digitonin has high affinity for cholesterol, thus low digitonin concentration will completely and selectively permeabilize plasma membrane (richer in cholesterol), compared to mitochondrial and endoplasmic reticulum membranes (low in cholesterol), so the latter are affected only at higher digitonin concentrations. It is known that molecular composition of plasma membrane differs between cell types and that modification in plasma membrane lipid composition occurs in tumor cells . Thus, every cell line requires previous optimization of adequate digitonin concentration, which is sufficient to permeabilize plasma membrane and which allows entrance of mitochondrial substrates and inhibitors, while keeping mitochondria intact and functional in their intracellular environment.
Zujovic T(1,2), Krako Jakovljevic N(1), Pavlovic K(1), Markovic I(3), Lalic NM(1)
- Clinic Endocrinology, Diabetes and Metabolic Diseases, CCS, Faculty of Medicine, Univ Belgrade - [email protected]
- Faculty of Biology, Univ Belgrade
- Inst Medical and Clinical Biochemistry, Faculty of Medicine, Univ Belgrade.
- Kojima K (1993) Molecular aspects of the plasma membrane in tumor cells. Nagoya J. Med. Sci. 56. 1 – 18.
- Krako N, Magnifico MC, Arese M, Meli G, Forte E, Lecci A, Manca A, Giuffrè A, Mastronicola D, Sarti P, Cattaneo A (2013) Characterization of mitochondrial dysfunction in the 7PA2 cell model of Alzheimer's disease. J Alzheimers Dis. 37(4):747-58.
- Gnaiger E, Kuznetsov AV, Lassnig B, Fuchs A, Reck M, Renner K, Stadlmann S, Rieger G, Margreiter R (1998) High resolution respirometry – optimum permeabilization of the cell membane by digitonin. . BioThermoKinetics in the Post Genomic Era, 89-95.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.
- Yang TT, Sinai P, Kain SR (1996) An Acid Phosphatase Assay for Quantifying the Growth of Adherent and Nonadherent Cells. Analytical Biochemistry 241(1): 103-108.
Preprints for Gentle Science