Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

SUIT-007 O2 ce-pce D030

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-007 O2 ce-pce D030



Abbreviation: Glutamate anaplerotic pathway

Reference: A pce: permeabilized cells - SUIT-007

SUIT number: D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama

O2k-Application: O2

SUITbrowser: Glutamine/glutamate

The SUIT-007 O2 pce D030 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample when glutamate is added alone, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyse the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and Electron transfer pathway coupling control states. In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.

Communicated by Cardoso LH, Komlodi T and Gnaiger E (last update 2019-07-03)

Representative traces

D030 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states


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 REN ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1G GL (N) CI 1G
2D GP (N) CI 1G;2D
2c GcP (N) CI 1G;2D;2c
  • Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M GMP N CI 1G;2D;2c;3M
4U GME N CI 1G;2D;2c;3M;4U
5Ama ROX 1G;2D;2c;3M;4U;5Ama
  • 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).


Click to expand or collaps

Strengths and limitations

  • Protocol focused on the analysis of glutamate anaplerotic pathway/glutaminolysis in permeabilized cells (ce-pce).
+ The use of glutamate alone allows to analyze the glutamate-anaplerotic pathway control state.
+ Short duration of the experiment.
+ Glutamate is easier to prepare compared to pyruvate.
+ CIV activity can be measured after this protocol.
- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity.
- This protocol does not address questions regarding the convergence of pathways at the Q-junction.

Compare SUIT protocols

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1G Glutamate (G)
2D ADP (D)
2c Cytochrome c (c)
3M Malate (M)
4U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry