Gnaiger 2008 POS: Difference between revisions
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== Figure legend == | == Figure legend == | ||
:::: Oxygen concentration and oxygen flux in intact cells with coupling control protocol. High-resolution respirometry (OROBOROS O2k with TIP2k) with parental hematopoietic 32D cells at 1.1ยท10<sup>6</sup> cells/cm<sup>3</sup> suspended in culture medium RPMI at 37 ยฐC. Replicate measurements in the two O2k-chambers (2 cm<sup>3</sup>). Superimposed plots of oxygen concentration [O<sub>2</sub>] and volume-specific oxygen flux, ''J''<sub>''V'',O2</sub>, calculated as the negative time derivative of oxygen concentration. [[ROUTINE respiration]] (''R'') is followed by inhibition of ATP synthase (manual titration of oligomycin, 2 ฮผgยทml<sup>-1</sup>) to induce nonphosphorylating [[LEAK respiration]] (''L''). Automatic titration of uncoupler (10 mM FCCP in the TIP2k) in steps of 0.1 ฮผl corresponding to a step increase in the final concentration of 0.5 ฮผM FCCP at intervals of 120 s. Maximum noncoupled flux (capacity of the electron transfer system, [[ETS capacity; state ''E'') is reached at 5.5 ฮผM FCCP. The ''L/E'' ratio is 0.10. Respiration is inhibited at higher [FCCP], unrelated to sample dilution (<1%). O2k-experiment 2005-04-09 EF-03, carried out by participants of an O2k-Workshop ([[Gnaiger 2014 MitoPathways]]). | :::: Oxygen concentration and oxygen flux in intact cells with coupling control protocol. High-resolution respirometry (OROBOROS O2k with TIP2k) with parental hematopoietic 32D cells at 1.1ยท10<sup>6</sup> cells/cm<sup>3</sup> suspended in culture medium RPMI at 37 ยฐC. Replicate measurements in the two O2k-chambers (2 cm<sup>3</sup>). Superimposed plots of oxygen concentration [O<sub>2</sub>] and volume-specific oxygen flux, ''J''<sub>''V'',O2</sub>, calculated as the negative time derivative of oxygen concentration. [[ROUTINE respiration]] (''R'') is followed by inhibition of ATP synthase (manual titration of oligomycin, 2 ฮผgยทml<sup>-1</sup>) to induce nonphosphorylating [[LEAK respiration]] (''L''). Automatic titration of uncoupler (10 mM FCCP in the TIP2k) in steps of 0.1 ฮผl corresponding to a step increase in the final concentration of 0.5 ฮผM FCCP at intervals of 120 s. Maximum noncoupled flux (capacity of the electron transfer system, [[ETS capacity; state ''E'') is reached at 5.5 ฮผM FCCP. The ''L/E'' ratio is 0.10. Respiration is inhibited at higher [FCCP], unrelated to sample dilution (<1%). O2k-experiment 2005-04-09 EF-03, carried out by participants of an O2k-Workshop ([[Gnaiger 2014 MitoPathways]]). | ||
== Further information == | |||
::::ยป [[MiPNet10.04 CellRespiration]] | |||
'''ยป''' [[MitoPedia: Respiratory states]] | '''ยป''' [[MitoPedia: Respiratory states]] | ||
[[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ETS capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] - [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]] | [[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ETS capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] - [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]] |
Revision as of 09:26, 9 October 2016
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52. |
Gnaiger E (2008) John Wiley
Abstract:
Several features distinguish high-resolution respirometry from traditional oxygraphs, combined in the new O2k (O2k, OROBOROS INSTRUMENTS, Innsbruck, Austria). The specifications are unique: the limit of detection of respiratory flux is 1 pmolโs-1โcm-3 (0.001 ยตMโs-1) and the limit of detection of oxygen concentration extends to 0.005 ยตM O2. For the non-specialist, the O2k provides robustness and reliability of instrumental performance. With small amounts of sample and correspondingly low respiratory flux per volume, the oxygen capacity of the system is exhausted slowly, allowing sufficient time to evaluate the stability of respiratory activity in each metabolic state and to permit complex titration regimes in intact cells or in permeabilized cells and tissues. To increase throughput in research with cell cultures and in the pharmacological arena, user-friendly features make it possible to apply several instruments in parallel, each O2k with two independent chambers.
โข O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse
Tissue;cell: Blood cells
Preparation: Intact cells, Permeabilized cells, Permeabilized tissue
Regulation: Coupling efficiency;uncoupling, Uncoupler 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, TIP2k, O2k-Protocol
O2k-Demo, O2k-Core
Figure legend
- Oxygen concentration and oxygen flux in intact cells with coupling control protocol. High-resolution respirometry (OROBOROS O2k with TIP2k) with parental hematopoietic 32D cells at 1.1ยท106 cells/cm3 suspended in culture medium RPMI at 37 ยฐC. Replicate measurements in the two O2k-chambers (2 cm3). Superimposed plots of oxygen concentration [O2] and volume-specific oxygen flux, JV,O2, calculated as the negative time derivative of oxygen concentration. ROUTINE respiration (R) is followed by inhibition of ATP synthase (manual titration of oligomycin, 2 ฮผgยทml-1) to induce nonphosphorylating LEAK respiration (L). Automatic titration of uncoupler (10 mM FCCP in the TIP2k) in steps of 0.1 ฮผl corresponding to a step increase in the final concentration of 0.5 ฮผM FCCP at intervals of 120 s. Maximum noncoupled flux (capacity of the electron transfer system, [[ETS capacity; state E) is reached at 5.5 ฮผM FCCP. The L/E ratio is 0.10. Respiration is inhibited at higher [FCCP], unrelated to sample dilution (<1%). O2k-experiment 2005-04-09 EF-03, carried out by participants of an O2k-Workshop (Gnaiger 2014 MitoPathways).