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SUIT-026 AmR ce-pce D087

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-026 AmR ce-pce D087



Abbreviation: RET

Reference: A - SUIT-026 - Protocol for the evaluation of ROS production by RET in permeabilized cells

SUIT number: D087_ce1;1Dig;1S;2Rot;3D;4Ama

O2k-Application: AmR

in preparation

SUIT-026 AmR ce-pce D087 has been designed to study the RET-initiated ROS production in (permeabilized cells (considered a type of mitochondrial preparation). Living cells are added to the chamber and then the plasma membrane is permeabilized with titration of digitonin. The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of rotenone provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I, thus leading to decreased ROS production. If rotenone would have been added before S titration, it would inhibit the RET. The titration of ADP at saturating concentrations to reach OXPHOS allows to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control. Addition of Ama leads to increased ROS production via inhibition of Complex III at the Qi site.

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.

Communicated by Cardoso LHD (last update 2022-09-05)

Representative traces

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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 ROX 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
2Rot SL(n) S CII 1S;2Rot
3D SP S CII 1S;2Rot;3D
4Ama ROX 1S;2Rot;3D;4Ama
  • 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

  • The conditions on which RET is observable in mitochondrial preparations are not physiological but it has been suggested that corresponds to some pathological states.
  • This protocol should be run in parallel with SUIT-026 O2 ce-pce D088 to have a respiratory control, since many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration.
+ Short duration of the experiment.
+ Rotenone provides an excellent control step for RET.
- This protocol does not provide information about all the coupling control states (LEAK respiration, OXPHOS and ET). However, it is possible to create a DLPU by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
- The addition of cytochrome c cannot be done in this SUIT protocol (SUIT-026 O2 ce-pce D088 must be run in parallel to assess the mt outer membrane integrity as a quality control of the sample).

Compare SUIT protocols

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)
2Rot Rotenone (Rot)
3D ADP (D)
4Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Fluorometry