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Difference between revisions of "SUIT-026"

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{{MitoPedia
{{MitoPedia
|abbr=RET
|abbr=RET
|description=[[File:SUIT-026 AmR mt D064.png|400px]]
|description=[[File:1S;2Rot;3D;4Ama.png|350px]]
|info='''A''' Short protcol to study RET-related H<sub>2</sub>O<sub>2</sub> production
|info='''A''' Short protocol to study [[RET]]-related H<sub>2</sub>O<sub>2</sub> production
}}
}}
::: '''[[SUIT protocol pattern]]:'''  1S;2Rot;3D;4Ama
::: '''[[SUIT protocol pattern]]:'''  1S;2Rot;3D;4Ama


SUIT-026 AmR mt D064 has been designed to  study the [[Reverse_electron_flow_from_CII_to_CI| RET]]-initiated ROS production, while SUIT-026 O2 mt D063 serves as a respiratory control protocol for SUIT-026 AmR mt D064 in [[Mitochondrial preparations| mitochondrial preparations]]: [[Isolated mitochondria|isolated mitochondria]] and [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|permeabilized cells]] (which are already permeabilized when they are added to the chamber). The protocol is focused on [[LEAK]] state for the [[S-pathway]] to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of [[rotenone]] provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I. The titration of ADP at saturating concentrations to reach [[OXPHOS]], allows us to harmonize this protocol with [[SUIT-006 O2 mt D022]] for quality control and a full assessment of the coupling control.  
SUIT-026 AmR mt D064 has been designed to  study the [[Reverse_electron_flow_from_CII_to_CI| reverse electron transfer]] (RET)-initiated [[ROS]] production, while SUIT-026 O2 mt D063 serves as a respiratory control protocol for SUIT-026 AmR mt D064 in [[Mitochondrial preparations| mitochondrial preparations]]: [[Isolated mitochondria|isolated mitochondria]] and [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|permeabilized cells]] (which are already permeabilized when they are added to the chamber). The protocol is focused on [[LEAK]] state for the [[S-pathway]] to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the [[Complex I]] occurs, promoting the production of ROS. The addition of [[rotenone]] provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I. The titration of ADP at saturating concentrations to reach [[OXPHOS]], allows us to harmonize this protocol with [[SUIT-006 O2 mt D022]] for quality control and a full assessment of the coupling control.  
 
According to our results, the H<sub>2</sub>O<sub>2</sub> production is linearly dependent on the O<sub>2</sub> concentration in MiR05-Kit, therefore, during the measurements the O<sub>2</sub> concentration has to be well-defined.




__TOC__
__TOC__
  Communicated by [[Komlodi T]] and [[Gnaiger E]] (last update 2019-06-26)
  Communicated by [[Komlodi T]], [[Cardoso LHD]] and [[Gnaiger E]] (last update 2020-09-02)


== Specific SUIT protocols ==
== Specific SUIT protocols ==
:[[File:SUIT-026 AmR mt D064.png|200px]]
 
:[[File:SUIT-026 AmR mt D064 O2 trace.png|500px]]
=== SUIT-026 AmR mt D064 ===
:[[File:SUIT-026 AmR mt D064 AmR trace.png|500px]]
[[File:1S;2Rot;3D;4Ama.png|300px]]
[[File:SUIT-026 AmR mt D064 O2 trace.png|500px]]
[[File:SUIT-026 AmR mt D064 AmR trace.png|500px]]
:[[SUIT-026 AmR mt D064]] to study RET-initiated H<sub>2</sub>O<sub>2</sub> production using isolated mtiochondria, tissue homogenate or permeabilized cells.
:[[SUIT-026 AmR mt D064]] to study RET-initiated H<sub>2</sub>O<sub>2</sub> production using isolated mtiochondria, tissue homogenate or permeabilized cells.
:[[File:SUIT-026 AmR mt D064.png|200px]]
 
:[[File:SUIT-026 O2 mt D063 O2 trace.png|400px]]
=== SUIT-026 O2 mt D063 ===
[[File:1S;2Rot;3D;3c;4Ama.png|300px]]
[[File:SUIT-026 O2 mt D063 O2 trace.png|400px]]
:[[SUIT-026 O2 mt D063]] serves as a control for [[SUIT-026 AmR mt D064]] in order to study the effect of the AmR assay on the respiration.
:[[SUIT-026 O2 mt D063]] serves as a control for [[SUIT-026 AmR mt D064]] in order to study the effect of the AmR assay on the respiration.
== Strengths and limitations ==
:::*  Many fluorescence dyes can inhibit components of the ET system and thus, affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye.
:::+ Short duration of the experiment.
:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS state.
:::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
:::- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.
:::- CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence.
== Compare SUIT protocols ==
:::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.




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== Strengths and limitations ==
== Strengths and limitations ==
:::* The conditions on which RET is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
:::* The conditions on which [https://wiki.oroboros.at/index.php/Reverse_electron_flow_from_CII_to_CI RET] is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
:::* The addition of cytochrome ''c'' is recommended for this SUIT protocol.
:::* Many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. Choose SUIT-026 O2 mt D063 as a control for SUIT-026 AmR mt D064.
:::* This protocol serves as a respiratory control for [[SUIT-026 AmR mt D064]].
:::+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.
:::+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.
:::+ Short protocol.
:::+ Short duration of the experiment.
:::- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
:::- CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence. [[SUIT-026 O2 mt D063]] can be used in parallel, with cytochrome ''c'' addition to assess the [[Cytochrome_c#Performance_and_data_analysis|mt outer membrane integrity]] as a quality control of the sample.
:::- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by adding an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
:::- Measurements with [[Amplex UltraRed]] assay cannot be carried out with [https://wiki.oroboros.at/index.php/Tissue_homogenate| liver homogenate].
 


== Compare SUIT protocols ==
== Compare SUIT protocols ==
::::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
::::* [[SUIT-006 O2 mt D022]]: CCP mtprep for S-pathway.
::::* [[SUIT-006 O2 mt D022]]: CCP mtprep for S-pathway.
::::* [[SUIT-026 AmR mt D064]]: Protocol to evaluate the RET production of ROS.





Revision as of 13:55, 2 September 2020


high-resolution terminology - matching measurements at high-resolution


SUIT-026

Description

1S;2Rot;3D;4Ama.png

Abbreviation: RET

Reference: A Short protocol to study RET-related H2O2 production

SUIT protocol pattern: 1S;2Rot;3D;4Ama

SUIT-026 AmR mt D064 has been designed to study the reverse electron transfer (RET)-initiated ROS production, while SUIT-026 O2 mt D063 serves as a respiratory control protocol for SUIT-026 AmR mt D064 in mitochondrial preparations: isolated mitochondria and tissue homogenate and permeabilized cells (which are already permeabilized when they are added to the chamber). The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of rotenone provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I. The titration of ADP at saturating concentrations to reach OXPHOS, allows us to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control. According to our results, the H2O2 production is linearly dependent on the O2 concentration in MiR05-Kit, therefore, during the measurements the O2 concentration has to be well-defined.


Communicated by Komlodi T, Cardoso LHD and Gnaiger E (last update 2020-09-02)

Specific SUIT protocols

SUIT-026 AmR mt D064

1S;2Rot;3D;4Ama.png SUIT-026 AmR mt D064 O2 trace.png SUIT-026 AmR mt D064 AmR trace.png

SUIT-026 AmR mt D064 to study RET-initiated H2O2 production using isolated mtiochondria, tissue homogenate or permeabilized cells.

SUIT-026 O2 mt D063

1S;2Rot;3D;3c;4Ama.png SUIT-026 O2 mt D063 O2 trace.png

SUIT-026 O2 mt D063 serves as a control for SUIT-026 AmR mt D064 in order to study the effect of the AmR assay on the respiration.


MitoPedia: SUIT

Steps and respiratory states

1S;2Rot;3D;4Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2Rot SL(n) S CII 1S;2Rot
3D SP S CII 1S;2Rot;3D
3c SP S CII 1S;2Rot;3D;3c
4Ama ROX 1S;2Rot;3D;3c;4Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • The conditions on which RET is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
  • Many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. Choose SUIT-026 O2 mt D063 as a control for SUIT-026 AmR mt D064.
+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.
+ Short duration of the experiment.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence. SUIT-026 O2 mt D063 can be used in parallel, with cytochrome c addition to assess the mt outer membrane integrity as a quality control of the sample.
- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a DLPU by adding an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.


Compare SUIT protocols

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
  • SUIT-006 O2 mt D022: CCP mtprep for S-pathway.


References

MitoPedia concepts: SUIT protocol, SUIT protocol, Recommended 


MitoPedia methods: Fluorometry