View source for SUIT-026

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

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]], [[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 respiration|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__
 Communicated by [[Timon-Gomez A]], [[Komlodi T]], [[Cardoso LHD]] and [[Gnaiger E]] (last update 2024-09-19)

== Specific SUIT protocols ==

=== SUIT-026 AmR mt D064 ===
[[File:Mt;1S;2Rot;3D;4Ama.png|300px]]
[[File:SUIT-026 AmR mt D064 O2 trace.png|400px]]
[[File:SUIT-026 AmR mt D064 AmR trace.png|400px]]
:[[SUIT-026 AmR mt D064]] to study RET-initiated H<sub>2</sub>O<sub>2</sub> production using [[mitochondrial preparations]] (isolated mtiochondria, tissue homogenate or permeabilized cells)

=== 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 respiration

=== SUIT-026 AmR ce-pce D087 ===
[[File:Ce1;1Dig;1S;2Rot;3D;4Ama.png|500px]]
[[File:SUIT-026 AmR ce-pce D087 O2 trace.png|400px]]
[[File:SUIT-026 AmR ce-pce D087 AmR trace.png|400px]]
:[[SUIT-026 AmR ce-pce D087]] to study RET-initiated H<sub>2</sub>O<sub>2</sub> production using cells, which are permeabilized in the chamber (ce-pce)

=== SUIT-026 AmR mt D077 ===
[[File:Mt;1S;2Rot;3D;4Ama.png|300px]]
[[File:SUIT-026 AmR mt D077 O2 trace.png|400px]]
[[File:SUIT-026 AmR mt D077 AmR trace.png|400px]]
:[[SUIT-026 AmR mt D077]] to study oxygen dependence of RET-initiated H<sub>2</sub>O<sub>2</sub> production using [[mitochondrial preparations]] (isolated mitochondria, tissue homogenate or permeabilized cells)

=== SUIT-026 O2 ce-pce D088 ===
[[File:ce1;1Dig;1S;2Rot;3D;3c;4Ama.png|400px]]
[[File:|400px]]
:[[SUIT-026 O2 ce-pce D088]] serves as a control for [[SUIT-026 AmR ce-pce D087]] in order to study the effect of the AmR assay on respiration.


{{Template:SUIT-026 O2 mt D063}}

== Strengths and limitations ==
:::* 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.
:::* 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 [[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 respiration, 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 ==
::::* [[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.


== References ==


{{MitoPedia concepts
|mitopedia concept=SUIT protocol, SUIT protocol, Recommended
}}
{{MitoPedia methods
|mitopedia method=Fluorometry
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics}}