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SUIT-006 NADH mt D084

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SUIT-006 NADH mt D084

Description

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Reference: A: protocol for simultaneous determination of O2 flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006

SUIT number: D084_1PGM;2D;3U;3anoxia;4Myx;5NADH

O2k-Application: NADH


Communicated by  Doerrier C (last update 2022-05-03) 


Representative traces

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MitoPedia: SUIT

Steps and respiratory states

Mt;1PGM;2D;3(Omy);4U;5Anox;6Myx;7Reox.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
mt REN mt
  • Respiration in the REN state is due to the presence of residual endogenous substrates.
  • In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
1PGM PGML(n) N CI 1PGM
2D PGMP N CI 1PGM;2D
(3Omy) PGML(Omy) N CI 1PGM;2D;(3Omy)
  • Omy addition is skipped in SUIT-006 NADH mt D084
  • NADH-linked substrates (type N-pathway to Q).
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U PGME N CI 1PGM;2D;(3Omy);4U
5Anox N CI 1PGM;2D;(3Omy);4U;5Anox
  • Anoxia, after the biological sample has consumed the O2 in the O2k-chamber, is used to detect the fully reduced NAD for calculation of the NAD redox states.
6Myx N CI 1PGM;2D;(3Omy);4U;5Anox;6Myx
  • The addition of myxothiazol after anoxia is a crucial step to detect the fully reduced NAD for calculation of the NAD redox state. Myxothiazol can induce a further reduction of NAD under anoxia.
7Reox ROX 1PGM;2D;(3Omy);4U;5Anox;6Myx;7Reox
  • The reoxygenation by opening the chamber in the presence of Myx allows for Rox-correction of the O2 fluxes.


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Strengths and limitations

Compare SUIT protocols

Chemicals and syringes

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

References