Reference: A Reference protocol RP1; mt: isolated mitochondria, tissue homogenate and permeabilized cells - SUIT-001
SUIT number: D001_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
The SUIT-001 O2 mt D001 protocol in combination with SUIT-002 O2 mt D005 is specially designed to provide 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 and tissues. SUIT-001 O2 mt D001 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 mt D001 can be extended with the CIV assay module.
Communicated by Doerrier C and Gnaiger E (last update 2019-03-27)
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|
- » Selected keywords in SUIT protocols
- Pathway control
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.
- + 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 mtprep, SUIT-001 and SUIT-002 harmonize in: SGpE (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.
- + Harmonization with many SUIT protocols (up to step 5S).
- + Oct is added in ET state to avoid uncoupler effect with fatty acids.
- + If NS- and FNS-pathways are similar (NSE ~ FNSE), the additivity index of N- and S-pathways in ET state can be evaluated.
- + This protocol can be extended with the Complex IV module.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- - Lengthy duration of the experiment.
- - We do not generally recommended the addition of permeabilized cells (already permeabilized), however we recommend to perform the permeabilization of the cell plasma membrane into the chambers (see SUIT-001 O2 ce-pce D003).
Compare SUIT protocols
- SUIT-001 O2 ce-pce D003: SUIT-001 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs into the chambers through digitonin addition. Therefore, SUIT-001 O2 ce-pce D003 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).
- SUIT-002 O2 mt D005 (RP2): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).
|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. Methods Mol Biol 1782:31-70.||Human|
MitoPedia methods: Respirometry