Difference between revisions of "Living cells"
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{{MitoPedia | {{MitoPedia | ||
|abbr=n.a. | |abbr=n.a. | ||
|description='''Intact cells''' are characterized by | |description='''Intact cells''' are characterized by an intact [[cell membrane]]. Cell viability should be >95% for various experimental investigations, including cell respirometry. In contrast, the cell membrane of intact cells can be permeabilized selectively by mild detergents ([[digitonin]]), to obtain the [[Mitochondrial preparations |mt-preparation]] of [[Permeabilized tissue or cells |permeabilized cells]] used for [[cell ergometry]]. | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods | ||
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== HRR and intact cells == | == HRR and intact cells == | ||
For | For details, see | ||
* [[ | * [[Gnaiger 2014 MitoPathways]] | ||
* [[O2k-Publications: | * Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. - [[Pesta 2012 Methods Mol Biol| »Bioblast link«]]. | ||
* [[Cell ergometry]] | |||
* [[O2k-Publications: Intact cells]] | |||
== | == Respiratoion medium == | ||
The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of cell culture | The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of [[cell culture media]] is the availability of substrates (e.g. glucose, glutamine), appropriate ionic composition for maintaining the cell membrane potential and intact signalling (particularly high [Ca<sup>2+</sup>]). Contidions during respiratory measurement can then be maintained close to cell culture conditions. | ||
Respiration of intact cells may be measured in respiration medium (e.g. [[MiR06]]) followed by permeabilization of the cell membrane by digitonin and applying complex [[SUIT]] (substrate-uncoupler-inhibitor titration) protocols. Measuring respiration of permeabilized cells, allowing direct access to the mitochondria, is not possible in cell culture media. These media contain high Ca<sup>2+</sup> concentrations, important for cell physiology, but damaging for mitochondria, which swell and disrupt. | Respiration of intact cells may be measured in mitochondrial respiration medium (e.g. [[MiR06]]) followed by permeabilization of the cell membrane by digitonin and applying complex [[SUIT]] (substrate-uncoupler-inhibitor titration) protocols. Measuring respiration of permeabilized cells, allowing direct access to the mitochondria, is not possible in cell culture media. These media contain high Ca<sup>2+</sup> concentrations, important for cell physiology, but damaging for mitochondria, which swell and disrupt. | ||
== Respiratory states == | == Respiratory states == | ||
[[ROUTINE]] and [[LEAK]] respiration, [[ETS]] capacity and [[ROX]] can be determined in intact cells (see [ | [[ROUTINE]] and [[LEAK]] respiration, [[ETS]] capacity and [[ROX]] can be determined in intact cells (see [[Gnaiger 2014 MitoPathways]]). These respiratory coupling states can be evaluated in the absence of external substrates on the basis of internal substrate stores (endogenous respiration). | ||
== Adherent cells== | == Adherent cells == | ||
The lab of [[US IL Springfield Brewer GJ|Gregory Brewer]] developed techniques for high-resolution respirometry with the OROBOROS-O2k of [[Talk:Brewer GJ|neuronal cells attached to a substrate]]: | The lab of [[US IL Springfield Brewer GJ|Gregory Brewer]] developed techniques for high-resolution respirometry with the OROBOROS-O2k of [[Talk:Brewer GJ|neuronal cells attached to a substrate]]: | ||
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* Minimum fluxes at 5 pmol.s<sup>-1</sup>.ml<sup>-1</sup> | * Minimum fluxes at 5 pmol.s<sup>-1</sup>.ml<sup>-1</sup> | ||
ROUTINE respiration may depend on cell density: | ROUTINE respiration per cell may depend on cell density: | ||
* | * Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - [[Steinlechner-Maran 1996 Am J Physiol Cell Physiol| »Bioblast link«]] | ||
=== Fibroblasts, HUVEC, thymocytes, lymphocytes === | === Fibroblasts, HUVEC, thymocytes, lymphocytes === | ||
1.0 million cells/ml is recommended for many cultured cells, including different cancer or immortalized cell lines. | 1.0 million cells/ml is recommended for many cultured cells, including different cancer or immortalized cell lines. A minimum of 0.1 million cells/ml is required. | ||
Revision as of 07:00, 3 September 2015
- high-resolution terminology - matching measurements at high-resolution
Living cells
Description
Intact cells are characterized by an intact cell membrane. Cell viability should be >95% for various experimental investigations, including cell respirometry. In contrast, the cell membrane of intact cells can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry.
Abbreviation: n.a.
MitoPedia methods:
Respirometry
MitoPedia topics: "Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property.
Respiratory state"Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property.
HRR and intact cells
For details, see
- Gnaiger 2014 MitoPathways
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. - »Bioblast link«.
- Cell ergometry
- O2k-Publications: Intact cells
Respiratoion medium
The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of cell culture media is the availability of substrates (e.g. glucose, glutamine), appropriate ionic composition for maintaining the cell membrane potential and intact signalling (particularly high [Ca2+]). Contidions during respiratory measurement can then be maintained close to cell culture conditions.
Respiration of intact cells may be measured in mitochondrial respiration medium (e.g. MiR06) followed by permeabilization of the cell membrane by digitonin and applying complex SUIT (substrate-uncoupler-inhibitor titration) protocols. Measuring respiration of permeabilized cells, allowing direct access to the mitochondria, is not possible in cell culture media. These media contain high Ca2+ concentrations, important for cell physiology, but damaging for mitochondria, which swell and disrupt.
Respiratory states
ROUTINE and LEAK respiration, ETS capacity and ROX can be determined in intact cells (see Gnaiger 2014 MitoPathways). These respiratory coupling states can be evaluated in the absence of external substrates on the basis of internal substrate stores (endogenous respiration).
Adherent cells
The lab of Gregory Brewer developed techniques for high-resolution respirometry with the OROBOROS-O2k of neuronal cells attached to a substrate:
- Jones TT, Brewer GJ (2009) Critical age-related loss of cofactors of neuron cytochrome c oxidase reversed by estrogen. Exp Neurology 215: 212-219
- Jones TT, Brewer GJ (2010) Age-related deficiencies in Complex I endogenous substrate availability and reserve capacity of Complex IV in cortical neuron electron transport. Biochim Biophys Acta Bioenergetics 1797: 167-176.
In most cases, adherent cells grown as a monolayer are detached from the culture plate (scrapping or trypsinizing), centrifuged and resuspended for HRR.
Appropriate cell density for HRR
The appropriate cell number is cell type and cell size dependent. The sample concentration should be high enough to get a reliable respiration even when mitochondria have a low respiratory activity. On the other hand, if respiration is too high, re-oxygenations have to be performed frequently disturbing the experimental course. As a general guideline:
- Maximum flux up to 100 to 150 pmol.s-1.ml-1
- Minimum fluxes at 5 pmol.s-1.ml-1
ROUTINE respiration per cell may depend on cell density:
- Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. - »Bioblast link«
Fibroblasts, HUVEC, thymocytes, lymphocytes
1.0 million cells/ml is recommended for many cultured cells, including different cancer or immortalized cell lines. A minimum of 0.1 million cells/ml is required.
Hepatocytes
Isolated hepatocytes are quite large, therefore, 0.1 million cells/ml can be applied.
