Difference between revisions of "SUIT-009 O2 mt D015"

Revision as of 13:14, 21 January 2019

high-resolution terminology - matching measurements at high-resolution

SUIT-009 O2 mt D015

Description

Abbreviation: NS(P)

Reference: A: SUIT-009 short protocol for respiration in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D015_1S;2D;3P;4Rot;5Ama

O2k-Application: O2

SUIT-category: NS(P)

SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot

1S;2D;3P;4Rot;5Ama-

SUIT-009 is a short protocol for simultaneous determination of O₂ flux on isolated mitochondria,tissue homogenate and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H₂O₂ production in the LEAK state. Addition of ADP increases S-OXPHOS capacity. Pyruvate (P) supports NADH-pathway (N). Antimycin A (Ama) inhibits Complex III (CIII) and results in the inhibition of O₂ flux. For H2O2-Application, choose SUIT-009 AmR mt D015; for O2 and H₂O₂ measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry

Strengths and limitations

Cytochrome C test can be performed in the S-OXPHOS state.

+Reasonable duration of the experiment.

@@ Line 4: / Line 4: @@
 |application=O2
 |description=[[File:1S;2D;3P;4Rot-.png|400px|SUIT9]]
-|info='''A''': '''[[SUIT-009]]''' short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in mitochondrial preparations (isolated mitochondria and tissue homogenate).
+|info='''A''': '''[[SUIT-009]]''' short protocol for respiration in mitochondrial preparations (isolated mitochondria and tissue homogenate).
 }}
 ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(P)
@@ Line 12: / Line 12: @@
 == 1S;2D;3P;4Rot;5Ama-==
-SUIT-009 is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]], and usually in the presence of ADP it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® assay (for determining H<sub>2</sub>O<sub>2</sub> production) is changing over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C.
+SUIT-009 is a short protocol for simultaneous determination of O<sub>2</sub> flux  on isolated mitochondria,tissue homogenate  and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP increases S-OXPHOS capacity. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]]. Antimycin A (Ama) inhibits Complex III (CIII) and results in the inhibition of O<sub>2</sub> flux.
-For O2-Application, choose [[SUIT-009 O2 mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.
+For H2O2-Application, choose [[SUIT-009 AmR mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.
 {{MitoPedia concepts
@@ Line 21: / Line 21: @@
 |mitopedia method=Respirometry
 }}
+== Strengths and limitations ==
+:::* Cytochrome C test can be performed in the S-OXPHOS state.
+::: +Reasonable duration of the experiment.