Difference between revisions of "Kuznetsov 2008 Nat Protoc"
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{{Publication | {{Publication | ||
|title=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- | |title=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. | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/18536644 PMID:18536644] | |info=[http://www.ncbi.nlm.nih.gov/pubmed/18536644 PMID: 18536644] | ||
|authors=Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS | |authors=Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS | ||
|year=2008 | |year=2008 | ||
|journal=Nat | |journal=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. | |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. | ||
|mipnetlab= | |mipnetlab=DE Magdeburg Gellerich FN, EE Tallinn Saks VA, FR Grenoble Saks VA, EE Tallinn Kaambre T | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
}} | }} | ||
==Corrections required== | == Corrections required == | ||
:: | ::* For published corrections, see p. 55 in [[Pesta 2012 Methods Mol Biol|Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58.]] | ||
: | * 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 MitoPathways|Gnaiger 2007]]; [[Pesta 2012 Methods Mol Biol]]). | |||
Latest revision as of 02:08, 9 October 2019
| 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
Labels:
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).
- 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).
