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Difference between revisions of "Attached cells"

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The Oroboros-O2k is principally designed to measure respiration in non-adherent, suspended samples, i.e. suspensions of cells or mitochondria, tissue homogenates or permeabilized fibers. However, successful efforts have been made to apply the Oroboros-O2k for measurements on adherent cells. Specifically, 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]]:
{{MitoPedia
* [[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]]
|abbr=
* [[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]].
|description=Many cell types are grown in culture as '''attached cells''', such as endothelial or neuronal cells in a monolayer.
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Additional general information is provided in the entry on [[intact cells]].
== HRR with attached cells ==
 
:::: The Oroboros O2k is principally designed to measure respiration in non-adherent, suspended samples, ''i.e.'', suspensions of cells or mitochondria, tissue homogenates, or permeabilized muscle fibers. However, successful efforts have been made to apply the O2k for measurements on adherent cells. Specifically, the lab of [[US IL Springfield Brewer GJ|Gregory Brewer]] developed techniques for high-resolution respirometry with the O2k using [[Talk:Brewer GJ|attached neuronal 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]].
 
::::* Further details: »[[Living cells]]
 
 
=== Fragile samples ===
 
:::: The rigorous stirring required for high-resolution respirometry causes problems for organisms / samples that are easily damaged by mechanical force, such as fish eggs or beads used to grow adherent cells.
 
:::: Presently an innovation is in preparation and will be available as a new product: a stainless steel mesh is used to maintain the sample separate from the stirrer compartment, without interfering with gentle mixing of the aqueous solution between the two compartments. Samples must be large enough not to pass through the 0.1 mm pore size of the mesh.
::::* Dias C, Lourenco CF, Barbosa RM, Laranjinha J, Ledo A (2018) Analysis of respiratory capacity in brain tissue preparations: high-resolution respirometry for intact hippocampal slices. Anal Biochem 551:43-50. - [[Dias_2018_Anal_Biochem |»Bioblast link«]] - Original publication on the tissue holder with brain slices.
 
=== Smaller stirring bars? ===
 
::::* '''Question:''' Would smaller stirring bars help to minimize damage (combined with lower stirring speed)? Would they be available?
 
::::* '''Answer:''' we are not sure if smaller stirring bars would help to avoid damage of fragile samples. In the early phase of the development of the O2k many different geometries and sizes of stirring bars were tested. In any case, commercially available Teflon (R) coated stirring bars cannot be used for high-resolution respirometry due to the high oxygen solubility of Teflon and the corresponding unacceptably high O2 background flux. The possibility of reducing the stirring speed can be evaluated by observing the noise and the [[stirrer test |response time] of the signal.
 
{{MitoPedia O2k and high-resolution respirometry
|mitopedia O2k and high-resolution respirometry=O2k-Open Support
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Latest revision as of 15:12, 1 March 2021


high-resolution terminology - matching measurements at high-resolution


Attached cells

Description

Many cell types are grown in culture as attached cells, such as endothelial or neuronal cells in a monolayer.


Template NextGen-O2k.jpg


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 



HRR with attached cells

The Oroboros O2k is principally designed to measure respiration in non-adherent, suspended samples, i.e., suspensions of cells or mitochondria, tissue homogenates, or permeabilized muscle fibers. However, successful efforts have been made to apply the O2k for measurements on adherent cells. Specifically, the lab of Gregory Brewer developed techniques for high-resolution respirometry with the O2k using attached neuronal cells:


Fragile samples

The rigorous stirring required for high-resolution respirometry causes problems for organisms / samples that are easily damaged by mechanical force, such as fish eggs or beads used to grow adherent cells.
Presently an innovation is in preparation and will be available as a new product: a stainless steel mesh is used to maintain the sample separate from the stirrer compartment, without interfering with gentle mixing of the aqueous solution between the two compartments. Samples must be large enough not to pass through the 0.1 mm pore size of the mesh.
  • Dias C, Lourenco CF, Barbosa RM, Laranjinha J, Ledo A (2018) Analysis of respiratory capacity in brain tissue preparations: high-resolution respirometry for intact hippocampal slices. Anal Biochem 551:43-50. - »Bioblast link« - Original publication on the tissue holder with brain slices.

Smaller stirring bars?

  • Question: Would smaller stirring bars help to minimize damage (combined with lower stirring speed)? Would they be available?
  • Answer: we are not sure if smaller stirring bars would help to avoid damage of fragile samples. In the early phase of the development of the O2k many different geometries and sizes of stirring bars were tested. In any case, commercially available Teflon (R) coated stirring bars cannot be used for high-resolution respirometry due to the high oxygen solubility of Teflon and the corresponding unacceptably high O2 background flux. The possibility of reducing the stirring speed can be evaluated by observing the noise and the [[stirrer test |response time] of the signal.


MitoPedia O2k and high-resolution respirometry: O2k-Open Support