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Kuznetsov 2008 Nat Protoc

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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: 965-976.

ยป 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.


โ€ข O2k-Network Lab: EE_Tallinn_Saks VA, FR_Grenoble_Saks VA


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Preparation: Permeabilized Cell or Tissue; Homogenate"Permeabilized Cell or Tissue; Homogenate" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 



HRR: Oxygraph-2k 


Corrections required

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

The protocol for permeabilized fibres lacks consideration on oxygen limitation encountered when incubations are performed at or below air saturation (Kuznetsov_1998_BTK; Gnaiger_2003_Adv. Exp. Med. Biol.). Protocols are restricted to simple substrate supply (separate CI- or CII-electron entry into the ETS), whereas full OXPHOS capacity can be obtained only with physiological CI+II substrate combinations (Pesta 2012 Methods Mol. Biol.).