Difference between revisions of "MiPNet08.09 CellRespiration"
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{{Publication | {{Publication | ||
|title=Gnaiger E, Renner K ( | |title=Gnaiger E, Renner K (2014) High-resolution respirometry and phosphorylation control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(08): 1-8. | ||
|info=[[Media:MiPNet08.09 CellRespiration.pdf|'''Open Access-MiPNet8.09.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions] [[Image:O2k-Protocols.jpg|right|150px|link=http://www.oroboros.at/?o2k-protocols|O2k-Protocols contents]] | |info=[[Media:MiPNet08.09 CellRespiration.pdf|'''Open Access-MiPNet8.09.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions] [[Image:O2k-Protocols.jpg|right|150px|link=http://www.oroboros.at/?o2k-protocols|O2k-Protocols contents]] | ||
|authors=OROBOROS | |authors=OROBOROS | ||
|year= | |year=2014 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract= | |abstract=An experiment on cellular respiration is reported from an Oxygraph-2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k chambers operated in parallel. Cellular ROUTINE respiration, JR, resulted in volume-specific oxygen consumption of 20 pmols-1ml-1. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (JL), and rotenone (residual oxygen consumption, JROX; after uncoupling) reduced respiration to 5 and 1 pmols1ml-1, while inducing the non-coupled state by FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmols-1ml-1. The ROUTINE flux control ratio, R/E, was 0.39 (uncoupling control ratio, UCR=E/R=2.6), and the LEAK flux control ratio, L/E, was 0.09 (E/L=12.0). This indicates tight coupling of OXPHOS, and a large respiratory excess capacity over ROUTINE respiration. The net ROUTINE flux control ratio, netR=(R-L)/E was 0.30, indicating that 30% of ETS capacity was activated for ATP production. | ||
An experiment on cellular respiration is reported from an Oxygraph-2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k chambers operated in parallel. Cellular ROUTINE respiration, JR, resulted in volume-specific oxygen consumption of 20 pmols-1ml-1. Oxygen concentration changed by merely 6.4 and 6.5 | |||
Automatic correction for instrumental background amounted to 13% for ROUTINE respiration, but to >50% and 180% for JL and JROX, respectively, illustrating the importance of real-time correction. The experiment illustrates the sensitivity and resproducibility of high-resolution respirometry with the OROBOROS Oxygraph-2k. Calibrations and routine corrections provide the basis of the high accuracy required for mitochondrial respiratory physiology. Analyses were performed real-time, combining high-resolution with instant diagnostic information. In this update graphs are presented illustrating some features of DatLab. | |||
:>> O2k-Protocols:[http://www.oroboros.at/?o2k-protocols Overall contents] | :>> O2k-Protocols:[http://www.oroboros.at/?o2k-protocols Overall contents] | ||
:>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | :>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | ||
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|additional=O2k-Demo | |additional=O2k-Demo | ||
}} | }} | ||
[[Image:MiPNet0809_FCR.jpg|center|width=50px|frame|The figure represents flux control ratios (FCR) normalized to state ETS (E) in chambers A and B. ROUTINE respiration in these cells is regulated at 0.39 of ETS capacity (R/E). Oligomycin (Omy) inhibits respiration to 0.08 ETS capacity (L/E).]] | |||
== Further information == | == Further information == | ||
>> Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and [MiPNet10.04 CellRespiration|MiPNet10.04]. | >> Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and [[MiPNet10.04 CellRespiration|MiPNet10.04]]. | ||
>> [http://www.oroboros.at/?protocols_cell_pcp Excel Templates and DatLab-Demo Files] | >> [http://www.oroboros.at/?protocols_cell_pcp Excel Templates and DatLab-Demo Files] | ||
Revision as of 06:49, 17 February 2014
Gnaiger E, Renner K (2014) High-resolution respirometry and phosphorylation control protocol with intact cells: ROUTINE, LEAK, ETS, ROX. Mitochondr Physiol Network 08.09(08): 1-8. |
» Open Access-MiPNet8.09.pdf; Versions
OROBOROS (2014) Mitochondr Physiol Network
Abstract: An experiment on cellular respiration is reported from an Oxygraph-2k workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k chambers operated in parallel. Cellular ROUTINE respiration, JR, resulted in volume-specific oxygen consumption of 20 pmols-1ml-1. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (JL), and rotenone (residual oxygen consumption, JROX; after uncoupling) reduced respiration to 5 and 1 pmols1ml-1, while inducing the non-coupled state by FCCP revealed the capacity of the electron transfer system (ETS) at JE of 50 pmols-1ml-1. The ROUTINE flux control ratio, R/E, was 0.39 (uncoupling control ratio, UCR=E/R=2.6), and the LEAK flux control ratio, L/E, was 0.09 (E/L=12.0). This indicates tight coupling of OXPHOS, and a large respiratory excess capacity over ROUTINE respiration. The net ROUTINE flux control ratio, netR=(R-L)/E was 0.30, indicating that 30% of ETS capacity was activated for ATP production.
Automatic correction for instrumental background amounted to 13% for ROUTINE respiration, but to >50% and 180% for JL and JROX, respectively, illustrating the importance of real-time correction. The experiment illustrates the sensitivity and resproducibility of high-resolution respirometry with the OROBOROS Oxygraph-2k. Calibrations and routine corrections provide the basis of the high accuracy required for mitochondrial respiratory physiology. Analyses were performed real-time, combining high-resolution with instant diagnostic information. In this update graphs are presented illustrating some features of DatLab.
- >> O2k-Protocols:Overall contents
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
• O2k-Network Lab: AT_Innsbruck_OROBOROS
Labels: MiParea: Respiration, Instruments;methods
Organism: Human
Tissue;cell: Blood cells
Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
O2k-Demo
Further information
>> Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and MiPNet10.04.