Zujovic 2019 MiP2019

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Tijana Zujovic
Cell membrane permeabilization: digitonin optimization pitfalls.

Link: MiP2019

Zujovic T, Krako Jakovljevic N, Pavlovic K, Markovic I, Lalic NM (2019)

Event: MiP2019

COST Action MitoEAGLE

Respirometry reflects the function of mitochondria and cell metabolism. Prior to performing respirometry, optimal cell number for each cell type has to be determined. This step is important to obtain optimal oxygen fluxes in the sensitivity range of the measuring device. In order to be able to study mitochondrial respiration with unpermeable substrates, cells have to undergo a controlled membrane permeabilization to allow washing out of endogenous substrates and enable exogenous substrates entering intact mitochondria. This can be achieved by digitonin, a mild detergent that has high affinity to cholesterol.This enables the plasma membrane which is rich in cholesterol to be permeabilized, while leaving intact endoplasmic reticulum and mitochondrial membranes which are low in cholesterol. Cell types differ in plasma membrane composition [1]. Thus, it is important to determine precise concentration of digitonin for every cell type prior to perform respirometric measurements. The aim of this study was to determine optimal cell number and digitonin concentration for controlled membrane permeabilization in human hepatocellular carcinoma cells (Huh7) and mouse skeletal myoblasts (C2C12).

We used high resolution respirometry performed with O2k Oxygraph, Oroboros to analyse mitochondrial respiration in permeabilized Huh7 and C2C12 cells. A recommendation for optimal cell number in a chamber is that ROUTINE (respiration of intact cells) yields a volume-specific oxygen flux of about 20 pmol s-1 mL-1 or higher [2]. The protocol for digitonin optimization was done according to [3].

We found that for both, Huh7 and C2C12 cell lines, optimal cell number was 0.5 million cells per O2k chamber, having ROUTINE respiration 90.05±5.93 pmol s-1 10-6 cells in Huh7 and 81.03±21.93 pmol s-1 10-6 cells in C2C12, or expressed as oxygen fluxes 18.29±5.31 pmol s-1 mL-1 in Huh7 and 19.01±2.9 pmol s-1 mL-1 in C2C12. Results are presented as average±standard deviation.

Optimal digitonin concentrations are 3 µg/0.5 million Huh7 cells and 5 µg/0.5 million C2C12 myoblasts.

Even though optimal digitonin concentration is established for each cell type, sometimes there is a need for additional optimization within same cell line, depending on treatment or cell differentiation stage. Our results show that palmitate treated Huh7 cells need almost twice the amount of digitonin used to permeabilize untreated Huh7 cells [4]. In C2C12 cells palmitate did not change their optimal digitonin concentration, but we faced a problem with this cell line when permeabilizing them after differentiation. We observed that cell membrane integrity was disrupted already after cell trypsinization step, since they respire as permeabilized even in the absence of digitonin. Cytochrome c test showed that mitochondrial membrane was affected as well.

To conclude, the digitonin optimization step is an essential part of respirometric measurements. Treatment or differentiation stage may influence optimal digitonin concentrations, especially if it affects cell membrane properties.


Bioblast editor: Plangger M, Tindle-Solomon L O2k-Network Lab: RS Belgrade Lalic NM


Labels: MiParea: Respiration 


Organism: Human, Mouse  Tissue;cell: Skeletal muscle, Liver  Preparation: Permeabilized cells 



HRR: Oxygraph-2k 


Affiliations

Zujovic Tijana(1,2), Krako Jakovljevic N(1), Pavlovic K(1), Markovic I(3), Lalic NM(1)
  1. Clinic Endocrinology, Diabetes Metabolic Diseases, CCS, Fac Medicine
  2. Fac Biology
  3. Inst Medical Clinical Biochemistry, Fac Medicine; Univ Belgrade, Serbia. - zujovictijana@gmail.com

References

  1. Kojima K (1993) Molecular Aspects of the Plasma membrane in tumor cells. Med Sci 56:1-18.
  2. 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.
  3. 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 membrane by digitonin. BTK-COX 95:89–94.
  4. Zujovic T, 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.