Difference between revisions of "SUIT-001 O2 ce-pce D004"
Timon Alba (talk | contribs) |
|||
(30 intermediate revisions by 7 users not shown) | |||
Line 1: | Line 1: | ||
{{MitoPedia | {{MitoPedia | ||
|abbr=RP1 ce-pce blood | |||
|description=[[File:ce1;1Dig;1PM;2D;2c;3U;4G;5S;(6Oct);7Rot;8Gp;9Ama;10AsTm;11Azd.png|400px|SUIT-RP1 for PBMC and PLT]] | |||
|info='''A''' - '''[[SUIT-001]]''' - '''[[SUIT reference protocol]] Reference protocol RP1 for living PBMC and PLT cells (ce, ROUTINE respiration) and cells that are permeabilized in the chamber (pce)''' | |||
|application=O2 | |application=O2 | ||
| | |SUIT number=D004_ce1;1Dig;1PM;2D;2c;3U;4G;5S;(6Oct);7Rot;8Gp;9Ama;10AsTm;11Azd | ||
}} | }} | ||
: | {{Template:SUIT text D004}} | ||
__TOC__ | __TOC__ | ||
Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2020-09-03) | |||
== | == Representative traces == | ||
[[File:D004_O2_traces.png|600px]] | |||
{{Template:SUIT- | {{Template:SUIT-001_blood}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
Line 41: | Line 29: | ||
:::+ Linear coupling control (''L''-''P''-''E'') with N substrates (PM). | :::+ Linear coupling control (''L''-''P''-''E'') with N substrates (PM). | ||
:::+ Pathway control in ET state (N, NS, FNS, S and SGp pathways). | :::+ Pathway control in ET state (N, NS, FNS, S and SGp pathways). | ||
:::+ Harmonization with [[SUIT-002]] allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be | :::+ Harmonization with [[SUIT-002]] allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements. | ||
:::+ In blood cells, SUIT-001 and SUIT-002 harmonize in: [[ROUTINE]] (ce1), SGp<sub>''E''</sub> (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV. | :::+ In blood cells, SUIT-001 and SUIT-002 harmonize in: [[ROUTINE]] (ce1), SGp<sub>''E''</sub> (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV. | ||
:::+ Harmonization with many SUIT protocols (up to step 5S). | :::+ Harmonization with many SUIT protocols (up to step 5S). | ||
:::+ This protocol can be extended with the Complex IV module. | :::+ This protocol can be extended with the Complex IV module. | ||
:::+ The addition of non-permeabilized cells to the chambers provides additional information ([[ROUTINE]] respiration) which can not be obtained in [[SUIT-001 O2 mt D001]] where the cells added to the chambers are already permeabilized. | |||
:::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S<sub>''E''</sub>. | :::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S<sub>''E''</sub>. | ||
:::- Oct is not added in ET state. | :::- Oct is not added in ET state. | ||
:::- Lengthy duration of the experiment. | |||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* SUIT-001 O2 ce-pce D004 (SUIT-001 for permeabilized PBMC and PLT) differs from [[SUIT-001 O2 ce-pce D003]] (general SUIT-001 for permeabilized cells) in the absence and presence of octanoylcarnitine, respectively. | |||
::::* [[SUIT- | ::::* [[SUIT-002 O2 ce-pce D007a]] (RP2): Harmonized SUIT protocol for PBMC and PLT cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). | ||
::::* [[SUIT-001 O2 mt D001]]: SUIT-001 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on [[ROUTINE]] respiration of non-permeabilized cells (ce). | |||
::::* [[SUIT-004]]: Short version of RP1; without G, F and Gp. | ::::* [[SUIT-004]]: Short version of RP1; without G, F and Gp. | ||
::::* [[SUIT-006]]: Very short initial coupling control module. | ::::* [[SUIT-006]]: Very short initial coupling control module. | ||
== Chemicals and syringes == | |||
{{Template:Chemicals ce}} | |||
{{Template:SUIT-001 O2 ce-pce D004}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | |||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-001 O2 ce-pce D004]] [[Additional label::SUIT-001]] | |||
| ?Was published in year=Year | |||
| ?Has title=Reference | |||
| ?Mammal and model=Organism | |||
| ?Tissue and cell=Tissue;cell | |||
| format=broadtable | |||
| limit=5000 | |||
| offset=0 | |||
| sort=Was published in year | |||
| order=descending | |||
}} | |||
{{MitoPedia concepts | {{MitoPedia concepts | ||
Line 62: | Line 76: | ||
|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
}} | }} | ||
Latest revision as of 10:02, 22 January 2024
Description
Abbreviation: RP1 ce-pce blood
Reference: A - SUIT-001 - SUIT reference protocol Reference protocol RP1 for living PBMC and PLT cells (ce, ROUTINE respiration) and cells that are permeabilized in the chamber (pce)
SUIT number: D004_ce1;1Dig;1PM;2D;2c;3U;4G;5S;(6Oct);7Rot;8Gp;9Ama;10AsTm;11Azd
O2k-Application: O2
The SUIT-001 O2 ce-pce D004 protocol is the reference protocol 1 (RP1) specifically for PBMCs (Peripheral Blood Mononuclear Cells) and PLTs (platelets). The main difference between the RP1 for PBMCs and PLTs and RP1 for other mitochondrial preparations (see SUIT-001) is the absence of Oct in the case of blood cells.
The SUIT-001 O2 ce-pce D004 protocol in combination with SUIT-002 O2 ce-pce D007a is specially designed to provide a common reference for comparison of respiratory control of PBMC and platelets cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of species. SUIT-001 O2 ce-pce D004 gives information of ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 ce-pce D004 can be extended with the CIV assay module.
Communicated by Doerrier C and Gnaiger E (last update 2020-09-03)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | N | CI | PML or PM_L: Pyruvate & Malate, N-LEAK respiration, NL
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3U | PME | N | CI | 1PM;2D;3U
|
4G | PGME | N | CI | 1PM;2D;3U;4G
|
5S | PGMSE | NS | CI&II | 1PM;2D;3U;4G;5S
|
(6Oct) | 1PM;2D;3U;4G;5S;(6Oct)
| |||
7Rot | SE | S | CII | 1PM;2D;3U;4G;5S;(6Oct);7Rot
|
8Gp | SGpE | SGp | CII&GpDH | 1PM;2D;3U;4G;5S;(6Oct);7Rot;8Gp
|
9Ama | ROX |
|
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
- SUIT-001 in combination with SUIT-002 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
- 1. to obtain reference values.
- 2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.
- 3. to screen specific defects.
- SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- + Linear coupling control (L-P-E) with N substrates (PM).
- + Pathway control in ET state (N, NS, FNS, S and SGp pathways).
- + Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
- + In blood cells, SUIT-001 and SUIT-002 harmonize in: ROUTINE (ce1), SGpE (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.
- + Harmonization with many SUIT protocols (up to step 5S).
- + This protocol can be extended with the Complex IV module.
- + The addition of non-permeabilized cells to the chambers provides additional information (ROUTINE respiration) which can not be obtained in SUIT-001 O2 mt D001 where the cells added to the chambers are already permeabilized.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- - Oct is not added in ET state.
- - Lengthy duration of the experiment.
Compare SUIT protocols
- SUIT-001 O2 ce-pce D004 (SUIT-001 for permeabilized PBMC and PLT) differs from SUIT-001 O2 ce-pce D003 (general SUIT-001 for permeabilized cells) in the absence and presence of octanoylcarnitine, respectively.
- SUIT-002 O2 ce-pce D007a (RP2): Harmonized SUIT protocol for PBMC and PLT cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol).
- SUIT-001 O2 mt D001: SUIT-001 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on ROUTINE respiration of non-permeabilized cells (ce).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
4G | Glutamate (G) | |
5S | Succinate (S) | |
(6Oct) | Octanoylcarnitine (Oct) | Oct is skipped in PBMCs and PTLs |
7Rot | Rotenone (Rot) | |
8Gp | Glycerophosphate (Gp) | |
9Ama | Antimycin A (Ama) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
## AsTm | Ascorbate (As) and TMPD (Tm) | |
## Azd | Azide (Azd) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet21.06 SUIT RP | 2018-06-25 | SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | ||
Doerrier 2018 Methods Mol Biol | 2018 | Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros 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 |
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Respirometry