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
- SUIT-026 AmR mt D064 to study RET-initiated H2O2 production using isolated mtiochondria, tissue homogenate or permeabilized cells.
SUIT-026 O2 mt D063
- 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
- SUIT-026 AmR ce-pce D087 to study RET-initiated H2O2 production using cells, which are permeabilized in the chamber.
SUIT-026 AmR mt D077
- SUIT-026 AmR mt D077 to study oxygen dependence of RET-initiated H2O2 production using isolated mtiochondria, tissue homogenate or permeabilized cells.
Steps and respiratory states
|Comment - Events (E) and Marks (M)
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- Pathway control
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 respiration, 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.
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MitoPedia methods: Fluorometry