Gnaiger, E*
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Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.
- Klíčová slova
- MEDORRHINUM,
- MeSH
- alergie MeSH
- diagnóza MeSH
- dítě MeSH
- homeopatie MeSH
- lidé MeSH
- mladiství MeSH
- neurovývojové poruchy MeSH
- příznaky a symptomy MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. For harmonization of results in a common database, we compared respiration and H2O2 production of permeabilized HEK 293T cells measured in MiR05 (sucrose and K-lactobionate), Buffer Z (K-MES and KCl), MiR07 (combination of MiR05 and Buffer Z), and MiRK03 (KCl). Respiration in a simple substrate-uncoupler-inhibitor titration protocol was identical in MiR05, Buffer Z, and MiR07, whereas oxygen fluxes detected with MiRK03 were consistently lower in all coupling and electron transfer-pathway states. H2O2 production rates were comparable in all four media, while assay sensitivity was comparatively low with MiR05 and MiR07 and higher but declining over time in the other two media. Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.
- MeSH
- buněčné dýchání MeSH
- buněčné kultury přístrojové vybavení metody MeSH
- fluorescenční barviva chemie MeSH
- fluorometrie přístrojové vybavení metody MeSH
- HEK293 buňky MeSH
- kalibrace MeSH
- kultivační média chemie MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oxaziny chemie MeSH
- permeabilita buněčné membrány MeSH
- peroxid vodíku metabolismus MeSH
- pufry MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L-1 ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.
- Publikační typ
- časopisecké články MeSH
