Kuznetsov 2008 Nat Protoc
|Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965-76.|
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Nat Protoc
Abstract: Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.
• O2k-Network Lab: DE Magdeburg Gellerich FN, EE Tallinn Saks VA, FR Grenoble Saks VA, EE Tallinn Kaambre T
- Table 1: Respiration of rat liver homogenate, 30 °C is actually mechanically permeabilized pig liver measured at 37 °C (Kuznetsov_2002_Analyt Biochem).
- The protocol (Nat Protoc) for permeabilized fibres lacks consideration of oxygen limitation encountered when incubations are performed at or below air saturation (Kuznetsov_1998_BTK; Gnaiger_2003_Adv Exp Med Biol). For a discussion, see »Permeabilized muscle fibres.
- Protocols (Nat Protoc) are restricted to simple substrate supply (separate CI- or CII-electron entry into the ET-pathway), whereas full OXPHOS capacity can be obtained only with physiological CI+II substrate combinations (Gnaiger 2007; Pesta 2012 Methods Mol Biol).