Difference between revisions of "SUIT-003 O2 ce D038"

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SUIT-003 O2 ce D038

Description

Abbreviation: CCP-ce Crabtree_E

Reference: A Coupling-control protocol and Crabtree effect test -SUIT-003 complex cell coupling-control protocol

SUIT number: D038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama

O2k-Application: O2

SUIT-category: ceGlc

SUIT-003 O2 ce D038 (CCP-ce Crabtree(E)) is designed to study coupling control in living cells and the effect of glucose (or fructose; Crabtree effect) on ET capacity. This protocol allows us to omit oligomycin, therefore the LEAK-respiration. If the aim is to analyze only the Crabtree effect, it is recommended to skip the oligomycin step. If there is an interest in studying coupling control, oligomycin should be added to promote LEAK-respiration. To obtain more information about the Crabtree effect, it is ideal to use in parallel the protocol SUIT-003 O2 ce D037 (glucose addition in ROUTINE). SUIT-003 O2 ce D038 can be extended by the cell viability test and the Complex IV module.

Communicated by Komlodi T , Garcia-Souza LF, Cardoso LHD and Gnaiger E (last update 2019-04-03)

Representative traces

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.
(ce2Omy)	L(Omy)	ce1;(ce2Omy) Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. If the aim is to analyse only Crabtree effect, it is recommended to skip this step. However, if there is also interest in studying coupling control, Omy can be added at this point to reach LEAK.
ce3U	E	ce1;(ce2Omy);ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce3Glc	E	ce1;(ce2Omy);ce3U;ce3Glc Noncoupled electron transfer state, ET state, with ET capacity E. Glucose is added to test Crabtree effect.
ce3'U	E	ce1;(ce2Omy);ce3U;ce3Glc;ce3'U Noncoupled electron transfer state, ET state, with ET capacity E. Additional uncoupler titrations are performed to ensure reaching ET state after addition of glucose.
ce4Ama	ROX	ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama 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.

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

The SUIT-003 O2 ce D038 is a cell coupling-control protocol. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).
This protocol can be extended with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
CIV activity can also be measured after the cell viability test module.

+ Reasonable duration of the experiment.

+ To obtain more information about Crabtree effect, this protocol can be used in parallel with SUIT-003 O2 ce D037. In SUIT-003 O2 ce D037, the addition of glucose in the ROUTINE state can lead to increase in ATP production by glycolysis, which can indirectly decrease respiration. In SUIT-003 O2 ce D038, with addition of glucose in ET state, this effect is bypassed, and only the direct effect of glucose on the electron transfer system is tested.

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

Compare SUIT protocols

SUIT-003 O2 ce D037 can be used in parallel to obtain more information on Crabtree effect.
SUIT-003 O2 ce-pce D013 for assessment of coupling control, Crabtree effect, malate effect and cell viability test.
SUIT-003 O2 ce-pce D018 for assessment of coupling control, Crabtree effect and cell viability test.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
(ce2Omy)	Oligomycin (Omy)	This step can be skipped
ce3U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
ce3Glc	Glucose (Glc)
ce3'U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
ce4Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=ceGlc
+|abbr=CCP-ce Crabtree_E
-|description=[[File:Ce1;ce2U;ce3Glc-.png|400px]]
+|description=[[File:Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png|350px]]
-|info='''A''' Coupling control protocol and Crabtree effect test -'''[[SUIT-003]]'''
+|info='''A''' Coupling-control protocol and Crabtree effect test -'''[[SUIT-003]]''' complex cell [[coupling-control protocol]]
 |application=O2
-|SUIT number=D038_ce1;ce2U;ce3Glc
+|SUIT number=D038_ce1;(ce2Omy);ce3U;ce3Glc;ce3'U;ce4Ama
 }}
-::: '''[[MitoPedia: SUIT]]'''
 ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ceGlc
-::: '''[[SUIT protocol pattern]]:''' complex cell [[coupling control protocol]] ce1;ce2U;ce3Glc
-SUIT-003 O2 ce D038 has been designed to study the [[coupling control state]] and respiration regulation by glucose (Crabtree effect) of intact cells on [[ET-capacity]]. To obtain more information about Crabtree effect, this protocol can be used in parallel with [[SUIT-003 O2 ce D037]]. SUIT-003 O2 ce D038 follows a modular approach and can be easily used in combination with the assessment of complex IV capacity and respirometric cell viability test.
+{{Template:SUIT text D038}}
+__TOC__
+ Communicated by [[Komlodi T ]], [[Garcia-Souza LF]], [[Cardoso LHD]] and [[Gnaiger E]] (last update 2019-04-03)
+== Representative traces ==
-__TOC__
+[[File:D038 O2 trace.png|600px]]
- Communicated by [[Garcia-Souza LF]] and [[Cardoso LH]] (last update 2019-03-26)
 {{Template:SUIT-003 O2 ce D038}}
 == Strengths and limitations ==
-:::* The SUIT-003 O2 ce D038 is a cell coupling control protocol. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>).
+:::* The SUIT-003 O2 ce D038 is a cell coupling-control protocol. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>).
 :::* This protocol can be extended with the cell viability test module (See CCV [[SUIT-003 O2 ce-pce D020]]).
 :::* CIV activity can also be measured after the cell viability test module.
 :::+ Reasonable duration of the experiment.
-:::+ To obtain more information about Crabtree effect, this protocol can be used in parallel with [[SUIT-003 O2 ce D037]]. In SUIT-003 O2 ce D037, the addition of glucose during ROUTINE state can lead to increase in ATP production by glycolysis, which can indirectly decrease respiration. In SUIT-003 O2 ce D038, with addition of glucose in ET state, this effect is bypassed, and only the direct effect of glucose on the electron-transfer system is tested.
+:::+ To obtain more information about Crabtree effect, this protocol can be used in parallel with [[SUIT-003 O2 ce D037]]. In SUIT-003 O2 ce D037, the addition of glucose in the ROUTINE state can lead to increase in ATP production by glycolysis, which can indirectly decrease respiration. In SUIT-003 O2 ce D038, with addition of glucose in ET state, this effect is bypassed, and only the direct effect of glucose on the [[Electron transfer pathway|electron transfer system]] is tested.
-:::- Cell culture media with glucose (''e.g.'' DMEM: 25 µM Glucose) could hinder the cell viability test.
 :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
@@ Line 30: / Line 30: @@
 ::::* [[SUIT-003 O2 ce D037]] can be used in parallel to obtain more information on Crabtree effect.
-::::* [[SUIT-003 O2 ce-pce D013]] for assessment of coupling control, Crabtree effect and cell viability test.
+::::* [[SUIT-003 O2 ce-pce D013]] for assessment of coupling control, Crabtree effect, malate effect and cell viability test.
-::::* [[SUIT-003 O2 ce-pce D018]] for assessment of coupling control, Crabtree effect, malate effect and cell viability test.
+::::* [[SUIT-003 O2 ce-pce D018]] for assessment of coupling control, Crabtree effect and cell viability test.
+== Chemicals and syringes ==
+{{Template:Chemicals SUIT-003 D038}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
 == References ==