SUIT-010

high-resolution terminology - matching measurements at high-resolution

SUIT-010

Description

Abbreviation: Digitonin test

Reference: A: Optimization of digitonin concentration for pce

SUIT protocol pattern: ce1;ce2Rot;ce3S;ce4D;1Dig;1c

SUIT-010 is designed for the evaluation of optimum digitonin concentration for permeabilizing cells, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of the natural product digitonin. After inhibition of the endogenous ROUTINE respiration by rotenone, respiration of living cells is not stimulated by succinate and ADP. Subsequent step-wise digitonin titration yields gradual permeabilization of the cell membrane, indicated by the increase of respiration up to full permeabilization. Respiration is constant over optimal digitonin concentration and inhibited by over-titration of digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to add cytochrome c after the optimal digitonin concentration had 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, Cardoso LHD, Huete-Ortega M, Gnaiger E (last update 2024-09-19)

Specific SUIT protocols

SUIT-010 O2 ce-pce D008

SUIT-010 O2 ce-pce D008 is a protocol to test the optimum concentration of digitonin

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 stepwise titrations for every batch. The optimum effective digitonin concentration 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 living 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

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: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry