Difference between revisions of "SUIT-026 AmR mt D064"
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|abbr=RET | |abbr=RET | ||
|description=[[File:SUIT-026 AmR mt D064.png|400px]] | |description=[[File:SUIT-026 AmR mt D064.png|400px]] | ||
|info='''A''' - '''[[SUIT-026]]''' | |info='''A''' - '''[[SUIT-026]]''' - Protocol for the evaluation of ROS production by RET in mtprep | ||
|application=AmR | |application=AmR | ||
|SUIT number=D064_1S;2Rot;3D;4Ama | |SUIT number=D064_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 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 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 leading to decreased ROS production. If rotenone would have been added before S titration, it had inhibited the RET. 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. Addition of [[Antimycin A|Ama]] leads to increased ROS production via inhibition of [[Complex III]] at the Qi site. | |||
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. In this protocol, we suggest not to go under ~100 µM O<sub>2</sub>. [[SUIT-018 AmR mt D041]] has been designed to study O<sub>2</sub> dependence of H<sub>2</sub>O<sub>2</sub> flux in [[mitochondrial preparations]]. | |||
__TOC__ | |||
Communicated by [[Komlodi T]], [[Gnaiger E]] (last update 2019-09-24) | |||
== Representative traces == | |||
[[File:SUIT-026 AmR mt D064 O2 trace.png|600px]] | |||
[[File:SUIT-026 AmR mt D064 AmR trace.png|600px]] | |||
{{Template:SUIT-026 AmR mt D064}} | {{Template:SUIT-026 AmR mt D064}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* The conditions | :::* 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. | ||
::: | :::* This protocol must be run in parallel with SUIT-026 O2 mt D063 to have a respiratory control, since many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration. | ||
:::+ Short | :::+ Short duration of the experiment. | ||
:::+ Rotenone provides an excellent control step for RET. | :::+ Rotenone provides an excellent control step for RET. | ||
:::- | :::- 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 additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state. | ||
:::- The addition of cytochrome ''c'' cannot be done in this SUIT protocol (SUIT-026 O2 mt D063 must be run in parallel to assess the [[Cytochrome_c#Performance_and_data_analysis|mt outer membrane integrity]] as a quality control of the sample). | |||
:::- 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- | ::::* [[SUIT-026 O2 mt D063]]: Respiratory control protocol. | ||
::::* [[SUIT- | ::::* [[SUIT-006 O2 mt D022]]: [[CCP]] with [[mtprep]] for S-pathway. | ||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals SUIT-026}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
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{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=SUIT protocol, SUIT | |mitopedia concept=SUIT protocol, SUIT A, Find | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods |
Latest revision as of 00:00, 13 April 2021
Description
Abbreviation: RET
Reference: A - SUIT-026 - Protocol for the evaluation of ROS production by RET in mtprep
SUIT number: D064_1S;2Rot;3D;4Ama
O2k-Application: AmR
SUIT-026 AmR mt D064 has been designed to study the RET-initiated ROS production 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 leading to decreased ROS production. If rotenone would have been added before S titration, it had inhibited the RET. 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. Addition of Ama leads to increased ROS production via inhibition of Complex III at the Qi site.
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. In this protocol, we suggest not to go under ~100 µM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.
Communicated by Komlodi T, Gnaiger E (last update 2019-09-24)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
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
|
4Ama | ROX | 1S;2Rot;3D;4Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
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.
- This protocol must be run in parallel with SUIT-026 O2 mt D063 to have a respiratory control, since many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration.
- + Short duration of the experiment.
- + Rotenone provides an excellent control step for RET.
- - This protocol does not provide information about all the coupling control states (LEAK respiration, OXPHOS and ET). However, it is possible to create a DLPU by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
- - The addition of cytochrome c cannot be done in this SUIT protocol (SUIT-026 O2 mt D063 must be run in parallel to assess the mt outer membrane integrity as a quality control of the sample).
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
Compare SUIT protocols
- SUIT-026 O2 mt D063: Respiratory control protocol.
- SUIT-006 O2 mt D022: CCP with mtprep for S-pathway.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
H2O2 | Hydrogen Peroxide (H2O2) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
0DTPA | DTPA | This step can be skipped |
0SOD | Superoxide Dismutase (SOD) | |
0HRP | Horseradish peroxidase (HRP) | |
0AmR | Amplex UltraRed (AmR) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1S | Succinate (S) | |
2Rot | Rotenone (Rot) | |
3D | ADP (D) | |
4Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry