Chowdhury 2000 Clin Chim Acta: Difference between revisions
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|abstract=Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9–22 independent cultures of amniocytes. | |abstract=Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9–22 independent cultures of amniocytes. | ||
|keywords=Prenatal diagnosis, Amniocytes, Oxidative phosphorylation, Respiratory-chain enzymes, Mitochondrial membrane potential, TMRM cytofluorometry | |keywords=Prenatal diagnosis, Amniocytes, Oxidative phosphorylation, Respiratory-chain enzymes, Mitochondrial membrane potential, TMRM cytofluorometry | ||
|mipnetlab=CZ Prague Houstek J, | |||
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Revision as of 13:57, 25 May 2012
| Chowdhury SK, Drahota Z, Floryk D, Calda P, Houstek J. (2000) Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes. Clin Chim Acta 298: 157-173. |
Chowdhury SK, Drahota Z, Floryk D, Calda P, Houstek J (2000) Clin Chim Acta
Abstract: Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9–22 independent cultures of amniocytes. • Keywords: Prenatal diagnosis, Amniocytes, Oxidative phosphorylation, Respiratory-chain enzymes, Mitochondrial membrane potential, TMRM cytofluorometry
• O2k-Network Lab: CZ Prague Houstek J
Labels:
Tissue;cell: Endothelial; Epithelial; Mesothelial Cell"Endothelial; Epithelial; Mesothelial Cell" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.
Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Oxygraph-2k
