Difference between revisions of "SUIT-009 O2 ce-pce D016"
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Communicated by [[Doerrier C]], [[Huete-Ortega M | Communicated by [[Doerrier C]], [[Huete-Ortega M]], [[Komlodi T]], [[Gnaiger E]] (last update 2019-06-06) | ||
{{Template:SUIT-009 O2 pce D016}} | {{Template:SUIT-009 O2 pce D016}} |
Latest revision as of 00:09, 13 April 2021
Description
Reference: B - SUIT-009 - short protocol for mt-respiration in ce: non-permeabilized cells, pce: permeabilized cells.
SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama
SUIT-009 O2 ce-pce D016 is a short protocol to investigate O2 flux in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended as a control experiment for SUIT-009 AmR ce-pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence 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.
Communicated by Doerrier C, Huete-Ortega M, Komlodi T, Gnaiger E (last update 2019-06-06)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | Dig | ce1;1Dig
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2D | SP | S | CII | 1S;2D
|
2c | ScP | S | CII | 1S;2D;2c
|
3P | NSP | NS | CI&II | 1S;2D;3P
|
4Rot | SP | S | CII | 1S;2D;3P;4Rot
|
5Ama | ROX | 1S;2D;3P;4Rot;5Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- 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
- 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 oxaloacetate on the succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration.
- It is a protocol to analyse the S- and NS-pathway linked respiration.
- For H2O2-Application, choose SUIT-009 AmR ce-pce D019.
- + Cytochrome c test can be performed in the S-OXPHOS state.
- + Short duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity.
- - In some cell lines e.g. HEK293T, P does not have 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 |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1S | Succinate (S) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3P | Pyruvate (P) | |
4Rot | Rotenone (Rot) | |
5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry