Difference between revisions of "SUIT-003 AmR ce D058"

Latest revision as of 16:06, 8 June 2020

high-resolution terminology - matching measurements at high-resolution

SUIT-003 AmR ce D058

Description

Abbreviation: AmR effect on ce

Reference: B: Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -SUIT-003

SUIT number: D058_ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: AmR

MitoPedia: SUIT - Protocol to analyse AmR effect on living cells respiration

SUIT protocol pattern: ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D058 has been designed to analyze the possible inhibitory effect of the Amplex UltraRed assay on the living cells coupling control state. Usually, the Superoxide dismutase and the Horseradish peroxidase do not show any inhibitory effect over the respiration. However, the Amplex UltraRed seems to inhibit ROUTINE respiration and the ET capacity at high concentrations. For this reason, the inhibitory effect of Amplex UltraRed should be tested with each type of sample used prior to the study of ROS production. SUIT-003 AmR ce D059 should be performed simultaneously as a carrier control titration.

Communicated by  Komlodi T, Garcia-Souza LF and Gnaiger E (last update 2019-06-24)

Steps and respiratory states

Step	State	Comment - Events (E) and Marks (M)
ce1	ROUTINE	ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce1SOD	ROUTINE	ce1;ce1SOD SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
ce1HRP	ROUTINE	ce1;ce1SOD;ce1HRP HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
ce1AmR	ROUTINE	ce1;ce1SOD;ce1HRP;ce1AmR AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.
ce2Omy	L_Omy	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Rot	ROX	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
ce5Ama	ROX	ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama 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).

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay over living cells respiration.
It is recommended to run this test with every different sample type used.
A carrier control titration should be run in parallel (SUIT-003 AmR ce D059).

+ Reasonable duration of the experiment.

- The concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET capacity.

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

SUIT-003 AmR ce D059 is a control protocol of to study the effect of the carrier titrations on the respiration in a parallel experiment.
SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin without the AmR assay

References

	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

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=ce
+|abbr=AmR effect on ce
-|description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]]
+|description=[[File:ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama.png|500px]]
 |info='''B:''' Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -'''[[SUIT-003]]'''
 |application=AmR
-|SUIT number=D058_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama
+|SUIT number=D058_ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama
 }}
-::: '''[[MitoPedia: SUIT]]'''
+::: '''[[MitoPedia: SUIT]]''' - Protocol to analyse AmR effect on living cells respiration
-::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ce
+::: '''[[SUIT protocol pattern]]:''' ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama
-::: '''[[SUIT protocol pattern]]:''' linear cell (ce) [[coupling control protocol]] ce1;ce2Omy;ce3U
-SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[coupling control state]] of intact cells. The different capacities are tested in the sequence [[OXPHOS]], [[LEAK]] and [[ET]]. Also, in order to study the [[LEAK-respiration]], we need to inhibit the [[phosphorylation system]] which in our protocol is achieved using [[oligomycin]]. However, the final concentration of [[oligomycin]] has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the [[Electron transfer-pathway|electron transfer system]], has to be studied to avoid an underestimation of the [[ET-capacity]]. [[Superoxide dismutase]] and [[Horseradish peroxidase]] do not inhibit respiration, however, [[Amplex UltraRed]] seems to block at higher concentration [[ROUTINE]] respiratoin and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type.
+{{Template:SUIT text D058}}
 __TOC__
-  Communicated by  [[Komlodi T]] and [[Gnaiger E]] (last update 2019-05-15)
+  Communicated by  [[Komlodi T]], [[Garcia-Souza LF]] and [[Gnaiger E]] (last update 2019-06-24)
 {{Template:SUIT-003 AmR ce D058}}
 == Strengths and limitations ==
-:::* This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay.
+:::* This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay over living cells respiration.
-:::* It is recommended to run this test with every differetn sample type.
+:::* It is recommended to run this test with every different sample type used.
+:::* A carrier control titration should be run in parallel ([[SUIT-003 AmR ce D059]]).
 :::+ Reasonable duration of the experiment.
-:::- The concentration of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET-capacity]].
+:::- The concentration of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET capacity]].
 :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
 == Compare SUIT protocols ==
+::::* [[SUIT-003 AmR ce D059]] is a control protocol of to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in a parallel experiment.
 ::::* [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin without the AmR assay
-::::* Protocols using AmR assay:
-:::::* [[SUIT-006 AmR mt D048]] to study simultaneously the O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.
-:::::*[[SUIT-009 AmR ce-pce D019]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )
-:::::* [[SUIT-009 AmR mt D021]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria and tissue homogenate
-:::::* [[SUIT-013 AmR ce D023]] for intact cells
-:::::*[[SUIT-018 AmR mt D031]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
-:::::* [[SUIT-018 AmR mt D040]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
-:::::* [[SUIT-018 AmR mt D041]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.
 == References ==
@@ Line 47: / Line 43: @@
 |order=descending
 }}
-[[File:1ce;2ceOmy;3ceU;4ceRot;5ceAma.jpg|right|200px]]
 {{MitoPedia concepts
-|mitopedia concept=SUIT protocol, SUIT B, Find
+|mitopedia concept=SUIT protocol, SUIT A, Find
 }}
 {{MitoPedia methods
 |mitopedia method=Respirometry
 }}
+{{MitoPedia O2k and high-resolution respirometry}}
+{{MitoPedia topics}}