MiPNet21.06 SUIT RP
 |
» [In progress]
Oroboros (2018-06-25) Mitochondr Physiol Network
Abstract: Doerrier C, Sumbalova Z, Krumschnabel G, Hiller E, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(01):1-12.
- » MitoPedia: SUIT reference protocol
- » Instrument: Oroboros O2k, O2k-Catalogue
• O2k-Network Lab: AT_Innsbruck_Oroboros
Labels: MiParea: Instruments;methods
Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria
Regulation: Substrate Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, Other combinations HRR: Oxygraph-2k, O2k-Protocol
MitoPathways, MitoFitPublication, SUIT-1 O2 mt D01, SUIT-1 O2 pfi D02, SUIT-1 O2 pce D03, SUIT-1 O2 pce D04, SUIT-2 O2 mt D05, SUIT-2 O2 pfi D06, SUIT-2 O2 pce D07
SUIT reference protocol RP1
SUIT states: 1-3PM(LPcE) 4PGM 5PGMS 6OctPGMS 7S 8SGp 9ROX 10Tm 11ROX
| Step | Respiratory state | Pathway control | Pathway to Q |
|---|---|---|---|
| 1PM | PM(L) | N | CI |
| 2D | PM(P) | N | CI |
| 2c | PM(P)c | N | CI |
| 3U | PM(E) | N | CI |
| 4G | PGM(E) | N | CI |
| 5S | PGMS(E) | NS | CI&II |
| 6Oct | OctPGMS(E) | FNS | FAO&CI&II |
| 7Rot | S(E) | S | CII |
| 8Gp | SGp(E) | SGp | CII&GpDH |
| 9Ama | ROX | ROX | |
| 10Tm | Tm(E) | CIV | CIV |
| 11Azd | ROX | ROX |
SUIT reference protocol RP2
SUIT states: 1ROX 2OctM(P) 2OctM(c) 3OctPM 4OctPGM 5OctPGMS 6OctPGMSGp 7E 8SGp 9ROX 10CIV 11ROX
| Step | Respiratory state | Pathway control | Pathway to Q |
|---|---|---|---|
| 1D | ROX | ROX | |
| 2M.1 | |||
| 3Oct | Oct(P) | (F) | FAO |
| 3c | OctM(P)c | F | FAO |
| 4M2 | OctM(P) | F | FAO |
| 5P | OctPM(P) | FN | FAO&CI |
| 6G | OctPGM(P) | FN | FAO&CI |
| 7S | OctPGMS(P) | FNS | FAO&CI&II |
| 8Gp | OctPGMSGp(P) | FNSGp | FAO&CI&II&GpDH |
| 9U | OctPGMSGp(E) | FNSGp | FAO&CI&II&GpDH |
| 10Rot | SGp(E) | SGp | CII&GpDH |
| 11Ama | ROX | ROX | |
| 11Tm | Tm(E) | CIV | CIV |
| 12Azd | ROX | ROX |
Harmonized SUIT protocols
RP1 and RP2 in mt-preparations
SUIT RP1 
SUIT RP2 
- SUIT RP1:1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
- SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2
RP1 and RP2 in cells
RP1 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see Digitonin). The use of intact cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address differentet Electron transfer-pathway states.
- SUIT RP1 in cells:ce1;1Dig;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
- SUIT RP2 in cells:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azdd
Harmonization between RP1 and RP2 in cells
RP1 and RP2 in PBMCs and PTLs
In PBMCs and PLTs the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMCs and PTLs
- SUIT RP1 for PBMCs and PLTs:1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
- SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2 in PBMC and PTL cells
RP1 and RP2 in cells
RP2 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see Digitonin). The use of intact cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address differentet Electron transfer-pathway statea.
- SUIT RP1 in cells: ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
- SUIT RP2 in cells: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azdd
Harmonization between RP1 and RP2 in cells
RP1 and RP2 in PBMCs and PTLs
In PBMCs and PLTs the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMCs and PTLs
- SUIT RP1 in PBMCs and PTLsSUIT RP1 for PBMCs and PLTs:1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
- SUIT RP2 PBMCs and PLTs: 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2 in PBMC and PTL cells
Experimental details
Work in progress: pre-publication protocol
Saturating ADP concentrations
- Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity. The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM.
SUIT-RP development
RP-T01
- RP1: Compared to RP1-T01, move Oct titration after S.
- RP2: Compared to RP2-T01, move U titration after Gp.
RP-T02
- RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct.
Cytochrome c test
- In protocol RP2, the cytochrome c test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect. On the other hand, at the low flux before titration of P, the sensitivity of the cytochrome c test may be low.
- ~ Gnaiger Erich 18:26, 23 January 2016 (CET)
Mark names
- Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.
- ~ Gnaiger Erich 16:50, 23 January 2016 (CET)
Pre-publication communication
- This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports. - Gnaiger Erich 03:23, 18 January 2016 (CET)
Experiments in progress
- The SUIT reference protocol is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMCs and platelets. - Gnaiger Erich 18:47, 19 January 2016 (CET)
- AT Innsbruck Gnaiger E and AT Innsbruck OROBOROS - MitoFit project.
- US NC Winston-Salem Molina AJA - UPBEAT project.
Further details
- » Introduction: Gnaiger 2014 MitoPathways
- » MitoPedia: SUIT - extended MitoPedia topic, May 2016
- Table of titrations » MiPNet09.12 O2k-Titrations
- » Definition: Substrate-uncoupler-inhibitor titration
- » Context: SUIT protocol library
- » Abbreviations: MitoPedia
