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SUIT-026

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SUIT-026

Description

SUIT-026 AmR mt D064.png

Abbreviation: RET

Reference: A

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

SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the SUIT-026 AmR mt D064 to study the RET-initiated ROS production in [[Mitochondrial preparations| 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.


Communicated by Komlodi T and Gnaiger E (last update 2019-06-26)

Specific SUIT protocols

SUIT-026 AmR mt D064.png
SUIT-026 AmR mt D064 to study RET-initiated H2O2 production using isolated mtiochondria, permeabilized cells or tissue homogenate.
SUIT-026 AmR mt D064.png
SUIT-026 O2 mt D063

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 H2O2 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 H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.


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


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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 addition of cytochrome c is recommended for this SUIT protocol.
  • 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.
+ Short protocol.
- 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 additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.

Compare SUIT protocols


References

MitoPedia concepts: SUIT protocol, SUIT protocol, Recommended 


MitoPedia methods: Fluorometry