Difference between revisions of "SUIT-003 AmR ce D059"

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SUIT-003 AmR ce D059

Description

Abbreviation: carrier control for AmR effect on living cells

Reference: B: Experimental control for SUIT-003 AmR ce D058

SUIT protocol pattern: ce1;ce2SOD;ce3HRP;ce4DMSO;ce5Omy;ce6U;ce7Rot;ce8Ama

SUIT-003 AmR ce D059 is a control protocol of SUIT-003 AmR ce D058 to study the effect of the carrier titrations on the respiration in a parallel experiment. This protocol should be run in parallel with SUIT-003 AmR ce D058 to compare the respiratory capacities and the Flux Control Ratios (FCR) in order to detect any inhibition of the respiratory capacities by the Amplex UltraRed or other components of the ROS assay.

Communicated by  Garcia-Souza LF, Komlodi T 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.
ce1H₂O	ROUTINE	ce1;ce1H₂O SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
ce1MiR05	ROUTINE	ce1;ce1H₂O;ce1MiR05 HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
ce1DMSO	ROUTINE	ce1;ce1H₂O;ce1MiR05;ce1DMSO AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.
ce2Omy	L_Omy	ce1;ce1H₂O;ce1MiR05;ce1DMSO;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E	ce1;ce1H₂O;ce1MiR05;ce1DMSO;ce2Omy;ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Rot	ROX	ce1;ce1H₂O;ce1MiR05;ce1DMSO;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;ce1H₂O;ce1MiR05;ce1DMSO;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 effect of the carrier titrations on the respiration in parallel to SUIT-003 AmR ce D058.
It is recommended to run this test with every different sample type used.

+ 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 D058: is a protocol to study the inhibitory effect of the AmR assay towards the respiration
SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin without the AmR assay

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=ce
+|abbr=carrier control for AmR effect on living cells
-|description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]]
+|description=[[File:1ce;2ceOmy;3ceU-.jpg|400px]]
-|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]]'''
+|info='''B:''' Experimental control for [[SUIT-003 AmR ce D058]]
-|application=AmR
-|SUIT number=D059_ce1;ce2SOD;ce3HRP;ce4DMSO;ce5Omy;ce6U;ce7Rot;ce8Ama
 }}
-::: '''[[MitoPedia: SUIT]]'''
+::: '''[[SUIT protocol pattern]]:''' ce1;ce2SOD;ce3HRP;ce4DMSO;ce5Omy;ce6U;ce7Rot;ce8Ama
-::: '''[[SUIT protocol pattern]]:''' ce1;ce2Omy;ce3U
-SUIT-003 AmR ce D059 is a control protocol of [[SUIT-003 AmR ce D058]] to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in a parallel experiment.
-SUIT-003 AmR ce D059 and SUIT-003 AmR ce D058 were designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[coupling control state]] of living 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]] respiration and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type. SUIT-003 AmR ce D058 shows the titration of the AmR assay, while SUIT-003 AmR ce D059 serves as a carrier control titration; these protocols should be run simultaneously.
+SUIT-003 AmR ce D059 is a control protocol of [[SUIT-003 AmR ce D058]] to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in a parallel experiment. This protocol should be run in parallel with [[SUIT-003 AmR ce D058]] to compare the respiratory capacities and the Flux Control Ratios ([[FCR]]) in order to detect any inhibition of the respiratory capacities by the [[Amplex UltraRed]] or other components of the ROS assay.
 __TOC__
+  Communicated by  [[Garcia-Souza LF]], [[Komlodi T]] and [[Gnaiger E]](last update 2019-06-24)
-  Communicated by  [[Garcia-Souza LF]], [[Komlodi T]](last update 2019-05-20)
@@ Line 24: / Line 17: @@
 == Strengths and limitations ==
 ::* This protocol has been designed to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in parallel to [[SUIT-003 AmR ce D058]].
-::* It is recommended to run this test with every different sample type.
+::* It is recommended to run this test with every different sample type used.
 ::+ 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]].
@@ Line 33: / Line 26: @@
 ::* [[SUIT-003 AmR ce D058]]: is a protocol to study the inhibitory effect of the AmR assay towards the respiration
 ::* [[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 ==