Since the neuron's membrane potential can sample its probabilitydistribution which determines the likelihood of a spike per unit time morerapidly than the timescale for changes in that probability distribution, our analyticmethods provide a reasonable description of the circuit's behavior (Fig. 4d).
13 The consistent and significant drop of TMRM fluorescence after treatments with the protonophore FCCP, with P < 0.001 and 0.2 < ΔΔ ψ < 0.8 for each study participant (Fig. 1), demonstrates the presence of detectable levels of mitochondrial membrane potential in all samples.
Therefore, the values of mitochondrial membrane potential from each sample were expressed as ratios of red fluorescence intensity over green fluorescence intensity.
As discussed previously, the smaller red/green ratios from liver mitochondria could reflect mitochondrial degradation due to the sample preparation procedure or loss of membrane potential over time, as these samples were analyzed after the muscle samples.
Briefly, cells were harvested, washed with PBS-T, and incubated with 3, 3'-diethyloxacarbocyanine iodide (DiOC2) for 30 min. The membrane potential of positive control samples was collapsed by the addition of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 min prior to the addition of DiOC2.
In a control sample, the membrane potential (Δψm) was dissipated by addition of 8 µM antimycin A, 0.5 µM valinomycin, and 2 µM oligomycin.
When TMRM was used to detect mitochondrial membrane potential, in each experiment one sample was treated with the protonophore FCCP (4 µM) as a positive control (full mitochondrial depolarization).
Within the day of blood sampling mitochondrial membrane potential was analyzed by the nonquenching TMRM method.
xi represents a sample in mV of membrane potential values.
