Gnaiger 2008 POS
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.
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, ET Pathway: ROX HRR: Oxygraph-2k, TIP2k, O2k-Protocol
O2k-Demo, O2k-Core, SUIT-003 O2 ce D009, SUIT-003, SUIT-006 General O2 pce D053
- 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 1.1·106 cells/cm3 suspended in culture medium RPMI at 37 °C. 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 μg·ml-1) to induce nonphosphorylating LEAK respiration (L). Automatic titration of uncoupler (10 mM FCCP in the TIP2k) in steps of 0.1 μl corresponding to a step increase in the final concentration of 0.5 μM FCCP at intervals of 120 s. Maximum noncoupled flux (capacity of the Electron transfer-pathway, ET-capacity; state E) is reached at 5.5 μM 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).
Coupling control protocol
- CCP: 1ce;2ceOmy;3ceU-
- 1ce: addition of intact cells (ce); measurement of ROUTINE respiration.
- 2ceOmy: Oligomycin is permeable through the plasma membrane of intact cells (ce); measurement of LEAK respiration.
- 3ceU: Uncoupler titration, uncouplers are permeable throgh the plasma membrane of intact cells (ce); measurement of ET-capacity.
- 4ceRotAma: Rotenone and antimycin A; measurement of residual oxygen consumption of intact cells (ce) after inhibition of CI and CIII.