Difference between revisions of "SUIT-018"

Revision as of 18:13, 24 January 2019

high-resolution terminology - matching measurements at high-resolution

SUIT-018

Abbreviation: NS(GM)

Reference: A »Versions

SUIT-category:NS(GM)

SUIT protocol pattern: linear 1GMS;2D;-

SUIT-018 AmR mt D031 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria and tissue homogenate.

	Year	Reference	Organism	Tissue;cell
MiPNet24.10 H2O2 flux analysis	2021-10-22	Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
MiPNet20.14 AmplexRed H2O2-production	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of H₂O₂ production with Amplex UltraRed.	Mouse	Heart
Komlodi 2018 Methods Mol Biol	2018	Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.	Human Mouse	Skeletal muscle HEK
Makrecka-Kuka 2015 Biomolecules	2015	Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319	Human Mouse	Heart Nervous system HEK
Krumschnabel 2015 Methods Mol Biol	2015	Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.	Mouse	Nervous system

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
0DTPA				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GMS	GMS_L(n)	NS	CI&CII	1GMS Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	GMS_P	NS	CI&CII	1GMS;2D Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Ama	ROX			1GMS;2D;3Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

+ Reasonable duration of the experiment.

+ Simple protocol to evaluate the oxygen dependence of H₂O₂ production.

- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.

- H₂O₂ production can be studied in two different states, LEAK and OXPHOS.

- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=NS(GM)
-|description=
+|description=[[File:SUIT-018 AmR mt D031.png|200px|SUIT-018]]
 |info='''A''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-018.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-018.pdf Versions]
 }}
 ::: '''[[Categories of SUIT protocols |SUIT-category]]:'''NS(GM)
-::: '''[[SUIT protocol pattern]]:''' linear 1GMS;2D;3Ama
+::: '''[[SUIT protocol pattern]]:''' linear 1GMS;2D;-
 __TOC__
@@ Line 13: / Line 13: @@
 == References ==
+{{#ask:[[Category:Publications]] [[Additional label::SUIT-018]] [[Additional label::SUIT-018 AmR mt D031]]  [[Instrument and method::O2k-Protocol]]
+| ?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
+}}
 {{Template:SUIT-018}}
 == Strengths and limitations ==
+:::+ Reasonable duration of the experiment.
+:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production.
+:::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
+:::- H<sub>2</sub>O<sub>2</sub> production can be studied in two different states, LEAK and OXPHOS.
+:::- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber