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Difference between revisions of "Template:SUIT text D060"

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In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate allows to obtain further information about the mitochondrial respiratory capacities in living cells. The plasma membrane-permeable compounds [[NV118/MitoKit-CII]] (Snv) and [[NV161/MitoKit-CII]] (Mnanv) require cleavage by esterases present in the cytosol to release respectively [[succinate]] and [[malonate]], which will happen therefore only in [[living cells]] with intact plasma membrane. The use of [[NV118/MitoKit-CII| Snv]] after rotenone provides information about the [[S-pathway]] in physiological conditions. Rotenone inhibits [[Complex I]] and consequently the [[TCA cycle]] and respiration in living cells, but intracellular release of succinate from the permeable compound may lead to stimulation of respiration. Afterwards, the addition of [[NV161/MitoKit-CII| Mnanv]] will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol allows to evaluate the [[coupling control state |coupling control]] of living cells. Respiratory capacities are tested in a sequence of coupling states: [[ROUTINE]] and [[ET]]. Optionally, to study [[LEAK-respiration]], the [[phosphorylation system]] might be inhibited by [[oligomycin]]. The final concentration of [[oligomycin]] has to be carefully optimized for various cell types, to minimize the inhibitory effect on the [[Electron transfer-pathway|electron transfer system]] which would lead to an underestimation of [[ET-capacity]].
In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate allows to obtain further information about the mitochondrial respiratory capacities in living cells. The plasma membrane-permeable compounds [[NV118/MitoKit-CII]] (Snv) and [[NV161/MitoKit-CII]] (Mnanv) require cleavage by esterases present in the cytosol to release respectively [[succinate]] and [[malonate]], which will happen therefore only in [[living cells]] with intact plasma membrane. The use of [[NV118/MitoKit-CII| Snv]] after rotenone provides information about the [[S-pathway]] in physiological conditions. Rotenone inhibits [[Complex I]] and consequently the [[TCA cycle]] and respiration in living cells, but intracellular release of succinate from the permeable compound may lead to stimulation of respiration. Afterwards, the addition of [[NV161/MitoKit-CII| Mnanv]] will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol allows to evaluate the [[coupling control state |coupling control]] of living cells. Respiratory capacities are tested in a sequence of coupling states: [[ROUTINE]] and [[Electron transfer pathway]]. Optionally, to study [[LEAK-respiration]], the [[phosphorylation system]] might be inhibited by [[oligomycin]]. The final concentration of [[oligomycin]] has to be carefully optimized for various cell types, to minimize the inhibitory effect on the [[Electron transfer-pathway|electron transfer system]] which would lead to an underestimation of [[ET-capacity]].

Revision as of 09:15, 3 June 2020

In SUIT-003 O2 ce D060 protocol, the use of permeable succinate and malonate allows to obtain further information about the mitochondrial respiratory capacities in living cells. The plasma membrane-permeable compounds NV118/MitoKit-CII (Snv) and NV161/MitoKit-CII (Mnanv) require cleavage by esterases present in the cytosol to release respectively succinate and malonate, which will happen therefore only in living cells with intact plasma membrane. The use of Snv after rotenone provides information about the S-pathway in physiological conditions. Rotenone inhibits Complex I and consequently the TCA cycle and respiration in living cells, but intracellular release of succinate from the permeable compound may lead to stimulation of respiration. Afterwards, the addition of Mnanv will counteract this effect. Moreover, the SUIT-003 O2 ce D060 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK-respiration, the phosphorylation system might be inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET-capacity.