From Bioblast
Description
Abbreviation: RET (respiratory control) of SUIT-026 AmR mt D064
Reference: B - SUIT-026 - Protocol for the respiratory control of SUIT-026 AmR mt D064
SUIT number: D063_1S;2Rot;3D;3c;4Ama
O2k-Application: O2
SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the SUIT-026 AmR mt D064 (protocol to study the RET-initiated ROS production in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells which are already permeabilized when they are added to the chamber).
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) |
---|---|---|---|---|
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
|
- 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
- This protocol serves as a respiratory control (since fluorescence dyes may inhibit components of the ET system) and quality control for the sample for SUIT-026 AmR mt D064.
- + Short protocol.
- + The addition of cytochrome c to assess the mt outer membrane integrity provides a quality control of the sample, which cannot be performed in the protocol with Amplex UltraRed.
- - 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
- SUIT-026 AmR mt D064: Protocol to evaluate the RET production of ROS.
- SUIT-006 O2 mt D022: CCP mtprep for S-pathway.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1S | Succinate (S) | |
2Rot | Rotenone (Rot) | |
3D | ADP (D) | |
3c | Cytochrome c (c) | |
4Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Fluorometry