Abbreviation: RP2 mt
SUIT number: D005_1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
The SUIT-002 O2 mt D005 protocol in combination with SUIT-001_O2_mt_D001 provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-002 O2 mt D005 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 O2 mt D005 can be extended with the CIV assay module.
Communicated by Doerrier C and Gnaiger E (last update 2019-09-24)
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
- SUIT-002 in combination with SUIT-001 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-002 allows the depletion of endogenous substrates with ADP (1D).
- + The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
- + F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
- + Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
- + Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
- + Harmonization with many SUIT protocols (up to step 7S).
- + In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
- + This protocol can be extended with the Complex IV module.
- - S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- - Lengthy duration of the experiment.
- - We do not generally recommended the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see SUIT-002 O2 ce-pce D007).
Compare SUIT protocols
- SUIT-001 O2 mt D001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).
- SUIT-002 O2 ce-pce D007: SUIT-002 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs in the chambers through digitonin addition. Therefore, SUIT-002 O2 ce-pce D007 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|3c||Cytochrome c (c)|
|9U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|11Ama||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.
|MiPNet21.06 SUIT RP||2018-06-25|
|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. https://doi.org/10.1007/978-1-4939-7831-1_3||Human|
MitoPedia methods: Respirometry