Difference between revisions of "SUIT-013"

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SUIT-013

Description

Abbreviation: ce

Reference: A »Versions

SUIT-category: ce

SUIT protocol pattern: linear

This protocol is ideal to study the oxygen dependence of H₂O₂ production in intact cells. SUIT-013 AmR ce D023 is ideal for demonstration of the oxygen dependence in baker´s yeast. For more details see: MiPNet18.06 Amplex-Yeast The respiration medium has to be choosen carefully, since the Amplex UltraRed assay leads to artefact formation with the components of the buffer.

Communicated by  Komlodi T  (last update 2019-01-22)

DLP applications

References

Steps and respiratory states

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)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.

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

+ Reasonable duration of the experiment.

+ Quick demonstration of the oxygen dependence of H₂O₂ production.

+ No externally added substrates are required with yeast.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry, Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=ce
-|description=
+|description=[[File:SUIT013 AmR ce D023.png|300px]]
 |info='''A''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-013.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-013.pdf Versions]
 }}
@@ Line 7: / Line 7: @@
 ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ce
 ::: '''[[SUIT protocol pattern]]:''' linear
+::: This protocol is ideal to study the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in intact cells. SUIT-013 AmR ce D023 is ideal for demonstration of the oxygen dependence in baker´s yeast. For more details see: [[MiPNet18.06 Amplex-Yeast]] The respiration medium has to be choosen carefully, since the [[Amplex UltraRed]] assay leads to artefact formation with the components of the buffer.
 __TOC__
   Communicated by  [[Komlodi T]]  (last update 2019-01-22)
 == DLP applications ==
+:::* [[SUIT-013 AmR ce D023]]
+:::* [[SUIT-013 AmR pce D###]]??
+:::* [[SUIT-013 AmR mt D###]]??
 == References ==
-{{#ask:[[Category:Publications]] [[Additional label::SUIT-013]]  [[Additional label::O2k-Protocol]]
+{{#ask:[[Category:Publications]]   [[Additional label::O2k-Protocol]] [[Additional label::SUIT-013]]
 | ?Was published in year=Year
 | ?Has title=Reference
-| ?Mammal and model=Organism
-| ?Tissue and cell=Tissue;cell
 | format=broadtable
 | limit=5000
@@ Line 29: / Line 30: @@
 == Strengths and limitations ==
-:::* A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
-:::* In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
-:::+ Contribution of N pathway to the NS can be checked after the addition of Rot.
 :::+ Reasonable duration of the experiment.
-:::+ This protocol can be extended with the Complex IV module.
+:::+ Quick demonstration of the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production.
-:::- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
+:::+ No externally added substrates are required with yeast.
-:::- Careful washing is required after the experiment to avoid carry-over of inhibitors.