Abstract
Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.
| Original language | English |
|---|---|
| Pages (from-to) | 328-335 |
| Number of pages | 8 |
| Journal | Mitochondrion |
| Volume | 12 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Mar 2012 |
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Keywords
- Micro-oxygraphy
- Mitochondrial disorders
- OXPHOS
- Spectrophotometry
ASJC Scopus subject areas
- Cell Biology
- Molecular Biology
- Molecular Medicine
Cite this
Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells. / Invernizzi, F.; D'Amato, I.; Jensen, P. B.; Ravaglia, S.; Zeviani, M.; Tiranti, V.
In: Mitochondrion, Vol. 12, No. 2, 03.2012, p. 328-335.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells
AU - Invernizzi, F.
AU - D'Amato, I.
AU - Jensen, P. B.
AU - Ravaglia, S.
AU - Zeviani, M.
AU - Tiranti, V.
PY - 2012/3
Y1 - 2012/3
N2 - Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.
AB - Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.
KW - Micro-oxygraphy
KW - Mitochondrial disorders
KW - OXPHOS
KW - Spectrophotometry
UR - http://www.scopus.com/inward/record.url?scp=84858004724&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84858004724&partnerID=8YFLogxK
U2 - 10.1016/j.mito.2012.01.001
DO - 10.1016/j.mito.2012.01.001
M3 - Article
C2 - 22310368
AN - SCOPUS:84858004724
VL - 12
SP - 328
EP - 335
JO - Mitochondrion
JF - Mitochondrion
SN - 1567-7249
IS - 2
ER -