Difference between revisions of "RCR is low in isolated mitochondria"
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Revision as of 14:53, 10 May 2012
Problem
At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly if added at a concentration typically used for State 3 measurements (200-300 µM), which may stimulate flux below the ADP-saturated state of OXPHOS capacity. The slope increases sharply and returns to State 4 (LEAK), even before the plot of flux shows the true maximum, resulting in a low apparent RCR.
Tests and Solutions
- Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the Oxygraph Control window; see MiPNet12.06 O2k-Start). As the data recording interval is reduced, the flux appears more noisy, but represents transitions more accurately and reduces the apparent time-delay. At high flux per volume, the increased noise level presents no problem. Flux then shows a period of constant OXPHOS capacity rather than a sharp peak.
- Reduce the mitochondrial concentration, thus prolonging the duration of ADP-saturated respiration (OXPHOS capacity) over 120 seconds. If flux reaches a constant maximum value, then analysis is possible without application of signal deconvolution (standard plot of flux in DatLab).
- Otherwise, export the data into DatLab 2, and apply a time correction (signal deconvolution), as described by Gnaiger 2001 Respir Physiol.
- See also ‘Notes on Time resolution’.
