Reference: B - SUIT-009- short protocol for respiration in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells
SUIT number: D015_1S;2D;3P;4Rot;5Ama
SUIT-009 O2 mt D015 is a short protocol for determination of O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber). It is recommended as a control experiment for SUIT-009 AmR mt D021 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O2 flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone. The following Coupling-control protocol can be used to assess Succinate-linked respiration with Rotenone: SUIT-006 O2 mt D022.
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
Strengths and limitations
- We do not recommend this protocol to assess just respiration since rotenone is not added at the beginning and does not prevent the inhibitory effect of the oxaloacetate on the succinate dehydrogenase. This protocol should be used as a control to investigate the effect of the fluorescence dyes on the respiration.
- It is a protocol to investigate the S- and NS-pathway linked respiration.
- For H2O2-Application, use SUIT-009 AmR mt D021.
- + Cytochrome c test can be performed in the S-OXPHOS state.
- + Short protocol.
- + This protocol can be extended with the Complex IV module.
- + Contribution of N-pathway to the NS can be investigated after the addition of Rot.
- - A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity.
- - In some cell lines e.g. HEK293T, P has not any effect because of the downregulation of the N-linked pathway.
Compare SUIT protocols
- SUIT-006 O2 mt D022: to investigate the S(Rot)-linked respiration.
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|2c||Cytochrome c (c)|
|5Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
MitoPedia methods: Respirometry