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 Mammal;model: Mouse Tissue;cell: Blood cells Preparation: Intact cells, Permeabilized cells, Permeabilized tissue Regulation: Coupling efficiency;uncoupling, Uncoupler Coupling state: LEAK, ROUTINE, ETS Substrate state: ROX HRR: Oxygraph-2k, TIP2k, O2k-Protocol Additional: O2k-Demo, O2k-Core
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 system, [[ETS 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).