Description
Reference: A: protocol for simultaneous determination of O2 flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-033
SUIT number: D081_mt;1D.1;2PGM;3D2.5;4Anox;5Myx;6Reox
O2k-Application: NADH
SUIT-033 NADH mt D081 allows the study of mitochondrial respiration and NADH fluorescence in OXPHOS in the N-pathway. In the absence of ATPases in the sample, LEAK state can also be evaluated. In order to measure LEAK in the presence of ATPases, this protocol should be performed in parallel to SUIT-032 NADH mt D078.
After the addition of mitochondria in the absence of fuel substrates and ADP, Ren, respiration due to oxidation of endogenous substrates remaining after mitochondrial isolation is measured. For calibration of the fully oxidized NAD, this protocol contains a step with the titration of a small concentration of ADP, stimulating the depletion of the endogenous substrates and thus leading to accumulation of oxidized NAD. This protocol is used for cross-calibration of SUIT-032 NADH mt D078, where no low concentration of ADP is added before fuel substrates, allowing the measurement of LEAK respiration and NAD redox state.
Anoxia is reached by letting mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to an accumulation of reduced NAD. Under anoxia the complex III inhibitor myxothiazol is added and a further increase in the reduced NAD fraction can be observed. This step is then used for the calibration of the fully reduced NAD. At the end of the protocol, the reoxigenation of the chamber allows the measurement of Rox.
Communicated by Grings M, Cardoso Luiza HD (last update 2023-11-30)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
mt | REN | mt | ||
1D.1 | mt;1D.1
| |||
2PGM | PGM | N | CI | mt;1D.1;2PGM
|
3D2.5 | PGMP | N | CI | mt;1D.1;2PGM;3D2.5
|
4Anox | N | CI | mt;1D.1;2PGM;3D2.5;4Anox | |
5Myx | N | CI | mt;1D.1;2PGM;3D2.5;4Anox;5Myx
| |
6Reox | ROX | mt;1D.1;2PGM;3D2.5;4Anox;5Myx;6Reox |
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- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- SUIT-033 NADH mt D081 in combination with SUIT-032 NADH mt D078 provides NAD redox ratios in LEAK and OXPHOS states, measured simultaneously with respiration.
- + Reasonable duration of the experiment.
- + H2 gas from Oxia or N2/argon can be used to decrease O2 concentration to obtain anoxia faster.
- + Fully oxidized NAD can be obtained due to low ADP (0.1 μM) titration before the addition of fuel substrates in contrast to SUIT-032 NADH mt D078 or SUIT-006 NADH mt D084.
- - When ATPases are present in the sample, it is not possible to reach the LEAK state.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors. The addition of liver homogenate is recommended in the washing protocol to bind strong inhibitors.
- - The concentration of the oxidized and reduced NAD fraction cannot be determined.
- - Antimycin A and CCCP cannot be used due to the high chemical background effect on fluorescence.
- - Cytochrome c test cannot be performed during the protocol as it affects fluorescence. Cytochrome c test can be performed in the following protocol: SUIT-033 O2 mt D110.
- After myxothiazol titration, this protocol can be extended with the Complex IV assay.
Compare SUIT protocols
- SUIT-034 NADH mt D082: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol with uncoupler titrations and ET state evaluation.
- SUIT-032 NADH mt D078: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Without titration of low concentration of ADP (0.1 μM) for depletion of endogenous substrates. Therefore, it is possible to measure LEAK respiration, harmonizing it with SUIT-033 NADH mt D081 in the presence of ATPases in the sample.
- SUIT-033 O2 mt D110: Control protocol for respiration only, allowing for cytochrome c test.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D.1 | ADP (D) | |
2PGM | Pyruvate (P), Glutamate (G), and Malate (M) | |
3D2.5 | ADP (D) | |
4Anox | The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration. | |
5Myx | Myxothiazol | We do not recommend the use of any other inhibitor of complex III, like Antimycin A (Ama), due to the chemical background effect on fluorescence. |
6Reox | Reoxygenation can be performed by opening the chamber, see: Open chamber. |
- Suggested stock concentrations are shown in the specific DL-Protocol.