SUIT-018 AmR mt D040

high-resolution terminology - matching measurements at high-resolution

SUIT-018 AmR mt D040

Description

Abbreviation: NS(P)

Reference: A: SUIT-018 to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D040_1GMS;2D;3Ama

O2k-Application: AmR

SUIT-category: NS(GM)

SUIT protocol pattern: orthogonal 1GMS;2D;3Ama

This is a short protocol to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production on isolated mitochondria or tissue homogenate (except of liver homogenate). You can run the protocol 1.)under tissue normoxia (50-60 µM = [O₂]) SUIT-018 AmR mt D031, 2.) under tissue hyperoxia [> 120 µM = [O₂]] SUIT-018 AmR mt D040 or 3.) you can change the oxygen concentration during the experiment SUIT-018 AmR mt D041. The oxygen concentration in the chamber can be changed by the injection of nitrogen with a syringe. It is recommended to run both SUIT-018 AmR mt D031 and SUIT-018 AmR mt D040 in paralell or SUIT-018 AmR mt D041 alone in order to test the H₂O₂ production at different oxygen concentrations.

Communicated by [Komlodi T]]  (last update 2019-02-01)

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)
1GMS	GMS_L(n)	NS	CI&CII	1GMS Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	GMS_P	NS	CI&CII	1GMS;2D Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Ama	ROX			1GMS;2D;3Ama 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).

Strengths and limitations

+ Reasonable duration of the experiment.

+ Simple protocol to evaluate the oxygen dependence of H₂O₂ production.

- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.

- H₂O₂ production can be studied in two different states, LEAK and OXPHOS.

- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber

- CIV activity and Cytochrome c test cannot be performed together with the fluorescence.

Compare SUIT protocols

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry