Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Kuznetsov 2008 Nat Protoc

From Bioblast
Publications in the MiPMap
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.

» PMID: 18536644

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


HRR: Oxygraph-2k 

Corrections required

  • Table 1: Respiration of rat liver homogenate, 30 °C is actually mechanically permeabilized pig liver measured at 37 °C (Kuznetsov_2002_Analyt Biochem).
  • 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).