Eigentler 2012 Abstract Bioblast
|Eigentler A, Draxl A, Fontana-Ayoub M, Gnaiger E (2012) The PBI-Shredder - an auxiliary HRR-tool for the preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 17.12.|
Event: Bioblast 2012
Two well established methods of tissue preparation in studies of mitochondrial respiratory function are (1) isolated mitochondria and (2) permeabilized fibres. Working with permeabilized fibres compared to isolated mitochondria demonstrates some advantages as less tissue is required, the mitochondrial morphology is not fragmented and all mitochondrial populations are present [1; 2; 3]. On the other hand, homogenized tissue provides different advantages as the preparation is faster, no detergents like saponin are required, tissue heterogeneity may entail a statistical problem in application of fibres which is averaged in the homogenate preparation, oxygen limitation is reduced, smaller amounts of tissue are needed compared to isolated mitochondria and tissue homogenates are well suited for the study of mitochondrial respiration . Furthermore, tissue homogenates have various advantages in O2k-Fluorometry compared to permeabilized fibres (Pfi): (1) all preparations can be applied if the fluorophore is dissolved in the incubation medium (e.g. Amplex UltraRed) but the use of Pfi is not possible in the O2k-chamber if the fluorophore binds to the tissue or mitochondria (safranin); (2) hyperoxygenation is generally necessary when working with Pfi to avoid diffusion limitation and hypoxic conditions within the fibre, which is problematic for ROS production – oxygen limitation is less pronounced in tissue homogenate; (3) with Pfi, variability between chambers is high due to tissue heterogeneity, which restricts comparability when different protocols are applied in parallel in different O2k-chambers – with tissue homogenate, variability between chambers is restricted to instrumental reproducibility, the degree of homogenization and reproducibility of pipetting subsamples from the homogenate. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (e.g. heart, liver, brain) with high reproducibility of mitochondrial yield and mitochondrial function as evaluated with HRR.
- Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS (1998) Permeabilised cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184: 81-100.
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845.
- Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MM, Romestaing C, Hepple RT (2011) Mitochondrial structure and function are disrupted by standard isolation methods. PLoS One 6: e18317.
• Keywords: PBI-Shredder SG3
Organism: Mouse Tissue;cell: Heart, Nervous system, Liver Preparation: Homogenate
Andrea Eigentler 2, Anna Draxl 1, Mona Fontana-Ayoub 1, Erich Gnaiger 1,2
(1) Oroboros Instruments Corp, High Resolution Respirometry, Innsbruck, Austria
(2) Medical University of Innsbruck, Department of Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Laboratory, Innsbruck, Austria; Email: firstname.lastname@example.org
Contribution to K-Regio Project MitoCom Tyrol
Shown is the PBI Shredder SG3 for tissue homogenate preparation consisting of a heavy duty high torque SG3 driver with convertible handle, SG3 base with 3 position force setting lever (FSL), battery charger and two lithium ion batteries. The PBI Shredder is used with the Shredder-Tubes consisting of the Shredder Tube with Lysis Disk, serrated Shredder-Ram and Shredder-Screw Cap as displayed in the figure on the right side.