Stankova 2017 MiP2017
Mitochondria regulate critical biological processes and mitochondrial dysfunctions are associated with pathogenesis of many diseases. Mitochondria isolation procedures affect their morphology and may disturb some of their native properties. Moreover, a part of mitochondria is lost during isolation and the final preparation does not represent the total population of tissue mitochondria. Despite the known limitations, properly isolated mitochondria provide a powerful tool for bioenergetic studies. Usage of isolated mitochondria allows better control of the experimental conditions in comparison to intact/permeabilized cells or tissues. The choice of separation technique depends on the requirements regarding organelle purity, yield, activity and also the duration of the preparation. The most widely used method for mitochondria isolation remains differential centrifugation. The liver is a parenchymal organ containing high amount of relatively homogenous mitochondria population which allows usage of gentle homogenization procedure. On the other hand, high activities of proteases and phospholipases in the liver tissue could disturb mitochondria during isolation procedure. In the literature you can find a confusing diversity of protocols for isolation of liver mitochondria which differ above all in sedimentation forces and buffers composition. Utilization of a standardized procedure protocols allows achieving valid data and provides necessary tool for comparison of results from different laboratories.
Liver mitochondria were isolated by differential centrifugation from male Wistar rats as described previously . We tested the effect of composition of homogenization and isolation media (0.25 M sucrose vs 0.22 M mannitol + 0.07 M sucrose; pH 7.2 vs 7.4; addition of 0.5 mM EDTA vs EGTA and bovine serum albumin - 1g/l into the media) on mitochondrial respiration (Oroboros O2k-FluoRespirometer), calcium retention capacity (Calcium green), mitochondrial ROS production (DCFDA) and mitochondrial membrane potential (Safranine O). The yield, purity and the loss of mitochondria were monitored by measuring enzyme activities (succinate dehydrogenase, citrate synthase, glucose-6-phosphatase, alkaline phosphatase, acidic phosphatase, catalase), and total protein content (Bradford assay).
We did not observe any significant effect of the tested media on the yield, purity and activity of mitochondria. Mitochondria were well coupled and the integrity of the outer membrane, measured by an activation of respiration by cytochrome c, was similar in all cases. In the near future we would like to focus on the effect of centrifugation parameters on the yield and purity of liver mitochondria. With regard to results of these studies we would like to propose isolation protocols specific for in vitro experiments to obtain highly active mitochondria and specific for in vivo experiments to avoid the loss of defective mitochondria during the isolation procedure.
Labels: MiParea: Respiration
Organism: Rat Tissue;cell: Liver Preparation: Isolated mitochondria
- Staňková P(1), Endlicher R(1,2), Kučera O(1), Červinková Z(1)
- 1Dept Physiol
- 2Dept Anatomy; Fac Medicine Hradec Králové, Charles Univ, Prague, Czech Republic. – [email protected]
References and support
- Endlicher R, Drahota Z, Červinková Z (2016) In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine. Physiol Res 65:321-31.
- This work was supported by research program PROGRES Q40/02 and Inter-Cost LTC17044