Abbreviation: CCP mt PM - MgG
Reference: A: short coupling-control protocol for simultaneous determination of O2 flux and mitochondrial ATP production in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) -SUIT-006
SUIT number: D055_1PM;2D;3Cat;4U;5Ama
SUIT-006 MgG mt D055 is a protocol to investigate the O2 flux and mitochondrial ATP production using the fluorescent dye Magnesium Green. In this protocol, the NADH Electron transfer-pathway state can be analyzed in mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber). Addition of the substrates pyruvate & malate (PM) to the mitochondrial preparation leads to LEAK state, and further addition of ADP induces the OXPHOS state, in which respiration is coupled to ATP production. After ADP addition, the signal from MgG gradually decreases, reflecting the ANT-mediated exchange of ADP for ATP, the later one having higher affinity for Mg2+. This exchange is stopped by addition of the ANT inhibitor carboxyatractyloside (Cat), which leads to LEAK state. With the addition of uncoupler, the Electron transfer pathway state can also be assessed in this protocol.
In the DatLab software, SUIT-006 MgG mt D055 DLP file is currently provided for the category N(PM). For using this protocol with other substrate/inhibitor combinations (e.g. succinate and rotenone for Succinate pathway control state or PMS for NS-pathway control state), a personalized DLPU can be created.
Communicated by Cardoso LHD (last update 2019-04-02)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)
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- Pathway control
Strengths and limitations
- Before performing this protocol, a calibration with the fluorescent dye and the determination of the Kd of ADP and ATP to Mg2+ in the pertaining experimental conditions need to be done. For more information, see: Magnesium Green.
- The buffer for this assay must be chosen carefully. It can be performed in MiR05 prepared without MgCl2 (with addition of 1 mM MgCl2 during the calibration or prior to the experiment) or with the ANT buffer (Chinopoulos 2014 Methods Enzymol).
- Inhibitors of kinases and ATPases might be necessary depending on the mitochondrial preparation used, specially for permeabilized cells or tissue homogenates (Chinopoulos 2014 Methods Enzymol).
- For protocols with MgG, ADP must be prepared without MgCl2.
- + In this protocol, ATP production in OXPHOS can be assessed in parallel with respiration. The protocol also allows to analyse the coupling control (LEAK, OXPHOS and ET respiration) in the same ET-pathway control state.
- - This protocol does not include internal ET-pathway control steps.
- - Even though MgG seems not to inhibit respiration in isolated mitochondria, it is recommended to do an experimental control in the absence of the fluorescent dye to check for inhibitory effects. The following protocol can be used: SUIT-006 O2 mt D047.
- - The test for mitochondrial outer membrane integrity with cytochrome c cannot be performed together with MgG assays.
- The ATP synthase inhibitor oligomycin (Omy) can also be used, instead of carboxyatractyloside, to stop mitochondrial ATP production and induce LEAK state. However, high concentrations of Omy can decrease the ET capacity measured after addition of uncoupler, therefore the concentration must be tested.
Compare SUIT protocols
- SUIT-006 O2 mt D047 for coupling control with pyruvate and malate as substrates.
- SUIT-006 Fluo mt D034 coupling control with pyruvate and malate as substrates for analysing membrane potential and O2 flux.
- SUIT-006 AmR mt D048 coupling control with pyruvate and malate as substrates for analysing H2O2 flux and O2 flux.
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|Step||Chemical(s) and link(s)||Comments|
|1PM||Pyruvate (P) and Malate (M)||Can be substituted by other combination of substrates|
|2D||ADP (D) preparation for MgG assay||For this protocol, the ADP must be prepared without Mg2+ salts.|
|3Cat||Carboxyatractyloside (Cat)||Can be substituted for oligomycin|
|4U||SF6847||Can be substituted for other uncoupler|
|5Ama||Antimycin A (Ama)||Can be substituted for other CIII or CIV inhibitor|
- Suggested stock concentrations are shown in the specific DL-Protocol.
MitoPedia methods: Fluorometry