Description
Abbreviation: ceP,S
Reference: A Coupling control protocol and respirometric cell viability test (CCV protocol); ce: non-permeabilized cells, pce: permeabilized cells - SUIT-003
SUIT number: D020_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd
O2k-Application: O2
- MitoPedia: SUIT
- SUIT-category: ceP,S
- SUIT protocol pattern: complex cell coupling control protocol ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;1Dig;1c;2Ama;3AsTm;4Azd
SUIT-003 O2 ce-pce D020 has been designed to study the coupling control state and membrane permeability of intact cells. The different capacities are tested in the sequence OXPHOS, LEAK and ET. Also, in order to study the LEAK state, we need to inhibit the phosphorylation system which in our protocol is achieved using oligomycin. However, the final concentration of oligomycin has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the electron transfer system, has to be studied to avoid an underestimation of the ET-capacity. SUIT-003 O2 ce-pce D020 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity.
Communicated by Iglesias-Gonzalez J, Krumschnabel G, Doerrier C and Cardoso LH(last update 2019-03-27)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1P | ROUTINE | ce1;ce1P
| ||
ce2Omy | L(Omy) | ce1;ce1P;ce2Omy
| ||
ce3U | E | ce1;ce1P;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce5S | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S
| |||
1Dig* | SE | S | CII | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig
|
1c | SE | S | CII | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c
|
2Ama | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama
|
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
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- The SUIT-003 O2 ce-pce D020 is a cell coupling control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
- For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.
- When the cell viability test module is not incorporated into the experiment, complex III inhibitors can be used to reach Rox instead of rotenone.
- + Respirometric cell viability test correlates with other cell viability quantification methods.
- + With the extended module, S-ET capacity can be assessed after the performance of the coupling control protocol, as well as outer mt-membrane integrity with the addition of cytochrome c.
- + CIV activity can also be measured after the cell viability test module.
- + Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 and a half hours (cell coupling control, cell viability test module and CIV module).
- - Cells with a cell membrane succinate transporter are not suitable for the viability test.
- - Pathological profiles could hinder the cell viability test.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
- - The addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.
Compare SUIT protocols
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
Doerrier 2018 Methods Mol Biol | 2018 | Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, MΓ©szΓ‘ros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3 | Human Mouse Rat Saccharomyces cerevisiae | Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet |
Stadlmann 2006 Cell Biochem Biophys | 2006 | Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1Γ treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86. | Human | Endothelial;epithelial;mesothelial cell |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry