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SUIT-009 AmR ce-pce D019

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-009 AmR ce-pce D019



Abbreviation: H2O2 RET ce-pce S_L

Reference: B: short protocol for H2O2 (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009

SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama

SUIT-009 is a short protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports reverse electron flow from CII to CI (RET) with high H2O2 production in the LEAK state at low O2 flow, SL. Respiration is stimulated by ADP (OXPHOS capacity, SP), while H2O2 flux declines dramatically. Pyruvate (P) supports the NADH-pathway and thus the convergent NS-pathway with little further effect on H2O2 flux, NSP). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Qo level inhibiting respiration to the level of Rox, and generally increases the H2O2 flux. The sensitivity of the Amplex UltraRed® (AmR) changes over experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes, several H2O2 calibration steps are done during the experiment. The experimental control for O2 flux in the absence AmR is carried out with SUIT-009 O2 ce-pce D016. According to our results, the H2O2 production is linearly dependent on the O2 concentration in MiR05-Kit, therefore, during the measurements the O2 concentration has to be well-defined. In this protocol, we suggest not to go under ~100 µM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.

Communicated by Huete-Ortega M, Cardoso LHD, Doerrier C, Komlodi T, Gnaiger E (last update 2019-09-17)
MitoPedia: SUIT

Steps and respiratory states


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
  • 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.
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
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.
1Dig Dig ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
4Rot SP S CII 1S;2D;3P;4Rot
5Ama ROX
  • 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).


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Strengths and limitations

  • Information is obtained on respiration and ROS production in the S- and NS-pathway.
  • SUIT-009 O2 ce-pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
  • To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT-003 AmR ce D058 with AmR and SUIT-003 AmR ce D059 with carrier titration as a control. These two protocols should be done in parallel.
+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the protonmotive force, pmF.
+ Short duration of the experiment.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence assay.
- The H2O2 flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of cytosolic compounds.
- In the case of HEK293T cells, however, due to the high malate-linked anaplerotic capacity, malate is converted not only to oxaloacetate but also to pyruvate, such that addition of succinate alone supports NS-pathway capacity (no effect of pyruvate on OXPHOS capacity. Under these conditions, reverse electron transfer is inhibited in the LEAK state, which prevents excessively high H2O2 flow.

Compare SUIT protocols

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
  • SUIT-018 is a short protocol to study the oxygen dependence of O2 flux and H2O2 production in isolated mitochondria or tissue homogenate (except of liver).

Chemicals and syringes

Step Chemical(s) and link(s) Comments
H2O2 Hydrogen Peroxide (H2O2)
Step Chemical(s) and link(s) Comments
0DTPA DTPA This step can be skipped
0SOD Superoxide Dismutase (SOD)
0HRP Horseradish peroxidase (HRP)
0AmR Amplex UltraRed (AmR)
Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1S Succinate (S)
2D ADP (D)
3P Pyruvate (P)
4Rot Rotenone (Rot)
5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


MitoPedia concepts: SUIT protocol, SUIT B, Find 

MitoPedia methods: Fluorometry