SUIT-010 O2 ce-pce D008

high-resolution terminology - matching measurements at high-resolution

SUIT-010 O2 ce-pce D008

Description

Abbreviation: Dig titration-pce

Reference: A -SUIT-010 - Optimum digitonin concentration for pce

SUIT number: D008_ce1;ce2Rot;ce3S;ce4D;1Dig;1c

O2k-Application: O2

SUIT-010 O2 ce-pce D008 is designed for the evaluation of optimum digitonin concentration for selective cell membrane permeabilization, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of digitonin. After inhibition of the endogenous ROUTINE respiration by rotenone, respiration of intact cells is not stimulated by succinate and ADP. Subsequent stepwise digitonin titration yields gradual permeabilization of the cell membrane, indicating by the increase of respiration up to full permeabilization. Respiration is constant within optimal digitonin concentration and inhibited by over-titration with digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to titrate cytochrome c after the optimal digitonin concentration has been reached. In other SUIT protocols for permeabilized cells, the respiration of pce is stable, if the optimal digitonin concentration is used.

Communicated by Doerrier C, Komlodi T, Huete-Ortega M and Gnaiger E (last update 2024-09-19)

Representative traces

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.
ce2Rot	ROX			ce1;ce2Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
ce3S	S_L(n)	S	CII	ce1;ce2Rot;ce3S Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
ce4D	S_P	S	CII	ce1;ce2Rot;ce3S;ce4D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
1Dig	S_P	S	CII	ce1;ce2Rot;ce3S;ce4D;1Dig Succinate, S ( type S-pathway to Q). Multiple Digitonin titrations for complete plasma membrane permeabilization. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
1c	S_P	S	CII	ce1;ce2Rot;ce3S;ce4D;1Dig;1c Succinate, S ( type S-pathway to Q). 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.

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

Digitonin is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization can be determined with this protocol.
Plasma membrane permeabilization is faster than mitochondrial isolation.
This protocol might be used simultaneously for selecting optimum experimental cell concentrations.

Compare SUIT protocols

SUIT-003 have been designed to study the coupling control state of intact cells
SUIT-003 O2 ce D028 coupling-control protocol with succinate for permeability test
SUIT-006 protocol is a coupling-control protocol for mitochondrial preparations

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
ce2Rot	Rotenone (Rot)
ce3S	Succinate (S)
ce4D	ADP (D)
1Dig	Digitonin (Dig)
1c	Cytochrome c (c)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

	Year	Reference	Organism	Tissue;cell
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry

Labels:

SUIT-010