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Difference between revisions of "Living cells"

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{{MitoPedia
{{MitoPedia
|abbr=n.a.
|abbr=vce
|description='''Intact cells''' are characterized by a [[cell membrane]] that is impermeable to various dyes, such as [[trypan blue]].
|description=Cell viability in '''living cells''' should be >95 % for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane barrier function. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>). In contrast, the cell membrane can be permeabilized selectively by mild detergents ([[digitonin]]), to obtain the [[Mitochondrial preparations |mt-preparation]] of [[permeabilized cells]] used for [[cell ergometry]]. Living cells are frequently labelled as ''intact cells'' in the sense of the total cell count, but ''intact'' may suggest the alternative meaning of ''viable'' or unaffected by a disease or mitochondrial injury.
|info=[[BEC 2020.1]], [[MiPNet08.09 CellRespiration]]
}}
}}
{{MitoPedia methods
|mitopedia method=Respirometry
}}
{{MitoPedia topics
|mitopedia topic=Respiratory state
}}
== HRR and intact cells ==


For primary information, see  
== HRR and living cells ==
* [[Pesta 2012 Methods Mol Biol|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]].
::: For details, see  
* [[O2k-Publications:_Intact_Cell;_Cultured;_Primary]]
::::* [[Gnaiger 2014 MitoPathways]]
::::* Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - [[Doerrier 2018 Methods Mol Biol| »Bioblast link«]].
::::* [[Cell ergometry]]
::::* [[O2k-Publications: Living cells]]
 
 
== Respiration 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 signaling (particularly high [Ca<sup>2+</sup>]). Conditions during respiratory measurement can then be maintained close to cell culture conditions.
 
:::: Respiration of viable cells may be measured in mitochondrial respiration medium (e.g. [[MiR05]]) 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 ==
:::: [[ROUTINE]] and [[LEAK]] respiration, [[Electron transfer pathway]] capacity and [[ROX]] can be determined in viable cells (see [[Gnaiger 2014 MitoPathways]]). These respiratory coupling states can be evaluated (''1'') in the absence of external substrates on the basis of internal substrate stores (endogenous respiration), (''2'') in the presence of specific fuel substrates, or (''3'') in complex culture media.
 
 
== 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]]: [[Attached_cells|Attached cells]]
::::* [[Jones_2009_ExpNeurol|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_2010_Biochim_Biophys_Acta|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.


== Respiratory medium ==


The choice of respiratory medium depends on the scientific question and the applied protocol. The advantage of cell culture medium is the availability of substrates (e.g. glucose, glutamine), appropriate ionic composition for maintaining the cell membrane potential and intact signalling. Contidions during respiratory measurement can then be maintained close to cell culture conditions.
== 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<sup>-1</sup>.mL<sup>-1</sup>
::::* Minimum fluxes at 5 pmol.s<sup>-1</sup>.mL<sup>-1</sup>


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.
:::: 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«]]




== Respiratory states ==
=== Fibroblasts, HUVEC, thymocytes, lymphocytes ===
:::: 1.0 million cells•mL<sup>-1</sup> is recommended for many cultured cells, including different cancer or immortalized cell lines. A minimum of 0.1 million cells/mL is required.


[[ROUTINE]] and [[LEAK]] respiration, [[ETS]] capacity and [[ROX]] can be determined in intact cells (see [http://www.oroboros.at/?Gnaiger_2012_MitoPathways Gnaiger 2012 MitoPathways]). These respiratory coupling states can be evaluated in the absence of external substrates on the basis of internal substrate stores (endogenous respiration).  
=== Hepatocytes ===
:::: Isolated hepatocytes are quite large, therefore, <0.1 million cells/mL can be applied.


=== Human peripheral blood mononuclear cells ===
:::: 2.0 million cells•mL<sup>-1</sup> is recommended for isolated PBMC. For more information see: [[MiPNet21.17_BloodCellsIsolation|»MiPNet21.17«]]


== Adherent cells==
=== Human platelets ===
:::: 100 million cells•mL<sup>-1</sup> is recommended for isolated platelets. For more information see: [[MiPNet21.17_BloodCellsIsolation|»MiPNet21.17«]]


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]]:
== Cell viability assessment ==
* [[Jones_2009_ExpNeurol|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_2010_Biochim_Biophys_Acta|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.


:::: Viability assays are used to measure the proportion of viable cells after a potentially traumatic procedure, such as primary disaggregation, cell separation, or cryopreservation. Most viability tests rely on a breakdown in membrane integrity measured by the uptake of a dye to which the cell is normally impermeable (''e.g.'', Trypan Blue) or the release of a dye normally taken up and retained by viable cells (''e.g.'', acridine orange & propidium iodide).


== Appropriate cell density for HRR ==
=== Trypan blue ===


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:
:::: Trypan blue is a vital dye. The reactivity of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged.  Therefore, all the cells which exclude the dye are viable.
* Maximum flux up to 100 to 150 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:
=== Acridine orange & propidium iodide ===
* [[Steinlechner-Maran 1996 Am J Physiol Cell Physiol|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-C2061.]]


:::: Acridine orange is an intercalating dye that can permeate both live and dead cells. Acridine orange will stain all nucleated cells to generate green fluorescence. Propidium iodide can only enter dead cells with poor membrane integrity so it will stain all dead nucleated cells to generate red fluorescence. Cells stained with both acridine orange and propidium iodide fluoresce red due to quenching, so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.


=== Fibroblasts, HUVEC, thymocytes, lymphocytes ===


1.0 million cells/ml is recommended for many cultured cells, including different cancer or immortalized cell lines.
== [[SUITbrowser]] question: Cell viability test ==


:::: Plasma membrane intactness can be assessed by [[SUIT]] protocols with the use of substrates that are not cell membrane permeant. With further chemical permeabilization of the cells, it is possible to determine the respirometric viability index, assuming that both viable and dead cells contain functional mitochondria.
:::: The [https://suitbrowser.oroboros.at/ SUITbrowser] can be used to find SUIT protocols for testing cell viability and other research questions.


=== Hepatocytes ===
== References ==
{{#ask:[[Additional label::Living cells]]
| mainlabel=Bioblast link
|?Has title=Reference
|?Was published in year=Year
|format=broadtable
|limit=5000
|offset=0
|sort=Has title
|order=ascending
}}


Isolated hepatocytes are quite large, therefore, 0.1 million cells/ml can be applied.
{{MitoPedia concepts}}
{{MitoPedia methods
|mitopedia method=Respirometry
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics
|mitopedia topic=Sample preparation
}}

Revision as of 07:54, 29 June 2020


high-resolution terminology - matching measurements at high-resolution


Living cells

Description

Cell viability in living cells should be >95 % for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane barrier function. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the cell membrane can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry. Living cells are frequently labelled as intact cells in the sense of the total cell count, but intact may suggest the alternative meaning of viable or unaffected by a disease or mitochondrial injury.

Abbreviation: vce

Reference: BEC 2020.1, MiPNet08.09 CellRespiration


HRR and living cells

For details, see


Respiration 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 signaling (particularly high [Ca2+]). Conditions during respiratory measurement can then be maintained close to cell culture conditions.
Respiration of viable cells may be measured in mitochondrial respiration medium (e.g. MiR05) 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, Electron transfer pathway capacity and ROX can be determined in viable cells (see Gnaiger 2014 MitoPathways). These respiratory coupling states can be evaluated (1) in the absence of external substrates on the basis of internal substrate stores (endogenous respiration), (2) in the presence of specific fuel substrates, or (3) in complex culture media.


Adherent cells

The lab of Gregory Brewer developed techniques for high-resolution respirometry with the OROBOROS-O2k of neuronal cells attached to a substrate: Attached cells
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-1 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.

Human peripheral blood mononuclear cells

2.0 million cells•mL-1 is recommended for isolated PBMC. For more information see: »MiPNet21.17«

Human platelets

100 million cells•mL-1 is recommended for isolated platelets. For more information see: »MiPNet21.17«

Cell viability assessment

Viability assays are used to measure the proportion of viable cells after a potentially traumatic procedure, such as primary disaggregation, cell separation, or cryopreservation. Most viability tests rely on a breakdown in membrane integrity measured by the uptake of a dye to which the cell is normally impermeable (e.g., Trypan Blue) or the release of a dye normally taken up and retained by viable cells (e.g., acridine orange & propidium iodide).

Trypan blue

Trypan blue is a vital dye. The reactivity of trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells which exclude the dye are viable.

Acridine orange & propidium iodide

Acridine orange is an intercalating dye that can permeate both live and dead cells. Acridine orange will stain all nucleated cells to generate green fluorescence. Propidium iodide can only enter dead cells with poor membrane integrity so it will stain all dead nucleated cells to generate red fluorescence. Cells stained with both acridine orange and propidium iodide fluoresce red due to quenching, so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red.


SUITbrowser question: Cell viability test

Plasma membrane intactness can be assessed by SUIT protocols with the use of substrates that are not cell membrane permeant. With further chemical permeabilization of the cells, it is possible to determine the respirometric viability index, assuming that both viable and dead cells contain functional mitochondria.
The SUITbrowser can be used to find SUIT protocols for testing cell viability and other research questions.

References

Bioblast linkReferenceYear
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-00022020
Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v12020



MitoPedia methods: Respirometry 



MitoPedia topics: Sample preparation