The model constructed, as described in Methods, is based on the generally accepted Q-cycle mechanism of complex III operation and predicts that it can evolve to a one of two different steady states. The direction of complex III evolution depends on the initial state of the system. If initially it is in highly reduced state, characterized by high levels of ubiquinol and semiquinone, it remains in this state, as shows thick gray lines in Figures 2A and 2B. However, if the initial levels of reduction is lower, the system evolves to another steady state, the same for a variety of initial states, as Figures 2A and 2B show. Thus, the Q-cycle mechanism defines bistability of complex III operation: with the same parameters it could function in a mode characterized by either low or high SQ levels (low or high ROS producing states respectively).

Various factors could trigger complex III from one mode to another, specifically, the availability of succinate could be a triggering factor. Figure 3A shows the time course of the transition from low to high ROS producing steady state, induced by an increase in succinate concentration (eq (17)). The capacity for ROS production is reflected in the concentration of free radicals, which can directly interact with oxygen, such as the semiquinone radical (SQ) bound to the Q_o site of complex III [14],[20]. An increase in succinate supply, and respective reduction of free ubiquinone (Q) to ubiquinol (QH₂), switches the system from low ROS producing state, characterized by low levels of SQ, to the high ROS producing state, characterized by high levels of SQ. In accordance with mass conservation, reduction of Q to QH₂ results in the deficiency of Q; this decreases electron flow and promotes reduction of cytochrome b. The QH₂ bound at Q_o site can freely deliver its first electron to the cytochrome c₁, but cannot deliver the second electron to cytochrome b_L because latter is already reduced. As a result, highly active SQ radicals are accumulated.

The continuum of steady state SQ levels and electron flow dependent on succinate supply is shown in Figures 3B and 3C. If the system is initially in a low ROS producing state, it switches to the high ROS production when the succinate concentration reaches a certain threshold value as Figure 3B shows. However, if the succinate concentration decreases back when the system initially is in high ROS producing state, it does not follow the same pattern, as Figure 3C shows. This example demonstrates hysteresis in complex III behavior.

The value of transmembrane potential is essential for such hysteretic behavior. As Figure S2 shows, the region of bistability is clearly distinguishable at transmembrane potential of ∼200 mV, which corresponds to the state 4 of mitochondrial respiration. The fall down of transmembrane potential to below 150 mV (as could be when the addition of ADP switches mitochondria to the state 3 of respiration), switches complex III from high to low ROS producing state. At high transmembrane potential the region of bistability persists over a large variation of model parameters as the sensitivity analysis shown in Text S1 and Figures S3 and S4 indicates. A switch from one steady state to another one essentially redistributes the fractions of various redox states of the complex as Figure S5 shows.

The electron flux in high ROS producing steady state, being restricted by the deficiency of Q, is low; maintaining it requires low succinate supply and this steady state persists even if substrate supply decreases until it falls down below the minimal threshold, as displayed in Figure 3C. The reduction of molecular oxygen by semiquinone radicals transforms them into Q, producing an acceptor able to take electrons from b_h, and thus to activate the Q-cycle. Thus, high ROS production rate, in a way, is a means to return back to low ROS production. Even a low rate of such an electron leak to oxygen helps the system to revert to low ROS production (see sensitivity analysis presented in Text S1). Normally direct transfer to oxygen insignificantly contributes to the total electron flow, but in the case of extremely high complex II activity, if it reduces all available Q, the electron flow to complex IV would equalize the ROS production rate.

The predicted phenomenon of bistability explored in Figure 3 could be observed experimentally, as Figure 4A shows. The isolated rat brain mitochondria incubated with succinate (state 4 of respiration) are in a high ROS producing state (blue trace “ros”), however, temporal presence of ADP, which is rapidly transformed into ATP, switches them to a low ROS producing mode (red trace “ros,ADP”). When ADP is present, ATP synthesis lowers membrane potential (shown in reverse direction as measured by quenching of fluorescence, red trace “mp,ADP”). However, after complete transformation of the added ADP into ATP, mitochondria again come back to state 4 of respiration, the membrane potential increases to the same levels as that without ADP (blue trace “mp”), but ROS production remains to be much lower (red trace “ros,ADP”). ADP only switched respiratory chain from high to low ROS production state, which could be maintained under the same conditions of incubation. The addition of ATP to the medium had no effect on ROS production (blue trace “ros”). In this case ROS production was the same as in the absence of ATP (Figure S6).

Figure 4B shows two branches of steady state ROS production rates measured before addition of ADP (stars) and after its consumption (squares) (as Figure 4A explains). Solid lines generated by computer simulation show that our model has reproduced these experimental data after implementing the Michaelis' dependence of complex II activity on succinate concentration (eq (17)). Thus, isolated mitochondria show the predicted bistable behavior (Figure 3) and the model simulates the particular experimental data (Figure 4B). In these experiments 5 mM of pyruvate were present at variable concentrations of succinate to ensure that overall substrate supply is not a limiting factor. In parallel experiments it was shown that at different succinate concentrations membrane potential was invariable (not shown). It should be noted that the parameters used as characteristics of succinate oxidation (Vm, Km) in complex II, in fact characterize several processes including succinate transport. In our in vitro experiments the amount of succinate must be sufficiently high to maintain the rate of its transport needed for ubiquinone reduction. The intramitochondrial succinate concentration, which in vivo is produced in TCA cycle and which is sufficient to trigger high ROS production, could be much less than that necessary to be added externally. The actual parameters, which govern the oxidation of succinate produced in the matrix in vivo, could be different, and since phenomenologically the mechanism admits the forward and reverse switch between the steady states (Figure 3), it could be expected under some conditions, such as ischemia, which could induce several fold increase in intracellular succinate content and, as a consequence, increase in FADH₂ [21]. Another factor, which substitutes succinate in triggering ROS production in vivo, is reduction of ubiquinone by complex I, whose substrate, NADH, is accumulated during anoxia [21].

The lack of oxygen itself can induce a switch to high ROS production if initially respiratory chain is in a low ROS production mode as Figure 4A shows. For this simulation the electron flux from complex III to complex IV was described as Michaelis' function of oxygen concentration (eq (18)). Parameters were chosen so that oxygen availability limits the flux when oxygen concentration falls to about 0.1% O₂, (in accordance with [13],[22]). The decrease of oxygen availability at the excess of succinate results in a reduction of ubiquinone and cytochrome b, and, finally, an accumulation of SQ radicals. As Figure 5A shows, the accumulation of SQ radicals proceeds in conjunction with limiting the electron flow by the lack of oxygen. Apparently, the physiological response to mild hypoxia, which could be observed at 5% O₂ [23], is related with another mechanisms.

If after passing through nearly anoxic conditions oxygen availability increases, the system remains in a high ROS producing state. Thus, the functional organization of electron transport in complex III, which has the property of bistability, explains the apparent paradox of increased ROS production induced by anoxia and re-oxygenation.

Figure 5B shows that anoxia induces a switch to a high ROS producing mode in isolated brain mitochondria, which were switched to a low ROS producing mode by temporal presence of ADP. Experiment was started with an open cuvette and normal oxygen supply by an addition of mitochondria to the incubation medium containing 1 mM ADP. Low value of membrane potential is indicative of mitochondria being initially in state 3. Upon conversion of all present ADP into ATP, mitochondria transited into state 4; an increased membrane potential is indicative of that transition. During the first stage of experiment mitochondria released low, but measurable amounts of ROS. An anoxic state was achieved by bubbling of the cuvette with N₂ for 2–3 min and then closing it with a Teflon lid. Slow depolarization of mitochondria and their persistence in the depolarized state for the duration of closure indicated an achievement of anoxic state. Notably, the ROS signal stayed flat during that stage. At the third stage of the experiment, the resumption of oxygen supply was achieved by opening the cuvette and bubbling it with air. At this stage mitochondria regained membrane potential up to the initial level thus showing their metabolic integrity. Significantly higher than at initial normoxic stage amounts of ROS were released upon the resumption of oxygen supply.

Although the damaging effect of reoxygenation after anoxia is well known in general, presented here experimental data for the first time directly demonstrate anoxia-induced increase in ROS at the levels of isolated mitochondria.

While at constant oxygen concentration the levels of SQ at Qo site could be considered as a relative rate of ROS production, the change of oxygen concentration must also affect it. Figure 6 shows the prediction of ROS production rate when the oxygen concentration changes as Figure 5A shows. For the calculations of ROS production eq. (16) was used with various K_ROS. Figure 6 indicates that the increase in ROS production could be detectable despite the decrease of oxygen concentration, if K_ROS is in the range of Km for respiration. Increase of K_ROS results in the decreases ROS production before it starts to affect the respiration rate that induces increase of SQ. In this case the change of SQ would not be well detectable. Thus, if experimentally ROS production was found to increase under anoxic conditions [3], it means that K_ROS must be in the range of Km for respiration or lower.

The algorithms for automated construction of large systems of differential equations, which are presented in Methods, allowed us to simulate in detail the Q-cycle mechanism of complex III operation and to find that the property of bistability is inherited from this mechanism. Due to this property it can operate in a low or high ROS producing mode at the same values of parameters, which explains the difference in ROS generation by respiratory chain before and after an episode of anoxia. The presented experiments qualitatively confirm the existence of two different ROS producing steady states in isolated mitochondria at the same microenvironmental conditions and a possibility to switch between them by the temporal presence of ADP and/or episodes of anoxia.

The following biochemical events were found to underlie the transition from low to high ROS producing state: (i) ubiquinone pool is reduced to ubiquinol, (ii) lack of oxidized form (ubiquinone) restricts cytochrome b_H oxidation and results in b_H and b_L reduction, which, in turn, (iii) restricts the oxidation of semiquinone radicals, thus increasing their concentration. If substrate concentration is then decreased below a threshold, the remaining electron flow is sufficient to maintain this high ROS producing state and therefore low and high ROS producing states could exist at the same set of parameters.

The increase of ROS production in tissues caused by ischemia-reperfusion is a well known clinical phenomenon; it is of great importance in various systemic diseases and organ transplantation. However, it was not known, which system is responsible for the switch from normally low ROS producing state to high ROS production, and how such a switch proceeds. We predicted and experimentally confirmed that complex III could respond to anoxia-reoxygenation in the same manner as it was observed at tissue levels. This indicates that the revealed mechanism could constitute the main part of tissue response to anoxia-reoxygenation. It does not exclude, though, a possible contribution of other parts of respiratory chain and/or other parts of metabolic and signaling pathways as modifiers of the analyzed phenomenon or even as other individual sources of ROS; rather, our findings open a new direction in investigation of the molecular mechanisms of bistable behavior that underlies the signaling effect of anoxia and oxidative stress caused by reoxygenation. In living cells ischemia combines several factors affecting ROS production, such as reduction of electron transport chain, anoxia, converting ATP into ADP and AMP, and deamination of the latter resulting in decrease of cellular adenilate pool [24]. Various combinations of these factors could create various types of behavior. Understanding their underlying mechanisms will improve the clinical care of diseases related to anoxia and also the quality of organ transplantation.

The proposed method for kinetic description of multienzyme complexes inherited from the established method of automated equation construction, which we developed for a different area of investigation, namely the analysis of stable isotopic isomers distribution in the intermediates of central carbohydrate metabolism [17]–[19]. Thus, it is not restricted by an area of mitochondrial respiration and could be further modified for applications in different areas, for instant for the analysis of metabolic signalling, which requires a description of multiple states of receptor molecules. Using this method will allow further expanding the model, including in it the other parts of electron transport chain and central metabolism, which provides substrates for mitochondrial respiration, ion transport that defines transmembrane potential, etc. Thus, the proposed method of differential equations construction opens a new direction for most detailed mathematical analysis of complex bioenergetic systems.

The model simulates the reactions performed by complex III, which overall expression is:The two electrons taken from QH₂ then are transferred to cytochrome c and further to complex IV and oxygen in conjunction with translocation of four more protons. Although the presented model does not consider the details of this latter process, it accounts for it stoichiometry.

In the course of complex III reaction its core (consisted of cytochrome b with its two hemes, b_H and b_L, cytochrome c₁, and Fe-S containing center of Rieske protein) bind and dissosiate ubiquinone/ubiquinol either in the matrix (Qi) or cytosolic (Qo) side of the inner mitochondrial membrane. This binding of quinones produce four different species of the complex:(1) (2) (3) (4)The electron transporters consisting the species (1)–(4) could be either in reduced or oxidized state. Various combinations of redox states of transporters produce many forms of each specie. The concentrations of such forms, which are transformed one into another during the complex operation, are the variables of the model. All the concentrations calculated for different redox forms of the same specie are stored in a specific array. To facilitate the references to variables inside the program, the two redox states of each transporter represented either by the digit “0” for oxidized state or by “1” for the reduced one. For instance, the binary “0011” (or decimal “3”) with respect to the array of core (1) refers to the concentration of a specie with oxidized b_H and b_L and reduced c₁ and FeS. On the other hand, these digits together form a binary number, which signifies that the concentration of this redox form occupies third position (starting from 0) in this array. Thus, the program uses such integer number for two purposes: to designate a specific redox form and in the same time to refer to the place of its concentration in the array reserved for the concentrations of all possible redox forms.

The total number of redox forms for a specie is defined by the number of transporters constituting it, being 2ⁿ, where n is the number of transporters. In this way, core (1) has 16 different redox states varying from 0000 to 1111; species (2) and (3) have 64 states each, varying from 000000 to 111111, and, respectively, the specie (4) has 256 redox states. The program constructs differential equations for each of these redox states based on the algorithms that reflect the redox reactions between transporters in accordance with the known biochemical mechanism of electron transport in complex III. As is schematically shown in Figure 2, the model takes into account 12 types of reactions, nine of them constitute the proper Q-cycle and three others are just related with complex III activity: superoxid anion formation (10), QH₂ supply by succinate dehydrogenase, and cytocrome c₁ oxidation by cytochrome c.

The algorithms for automated calculation of reaction rates for each redox form participated in thece 12 types of reactions is described in details next.

Reaction 0 transports the first electron of QH₂ bound on the positive (p-, or cytosolic) side to Fe3+ of Rieske protein and releases two protons to the intermembrane space.(5)The program simulates this reaction for all redox states of complex III capable to perform it, i.e. for the forms with attached QH₂ at Qo site and oxidized Fe-S center of Rieske protein. In binary terminology described above for species b_H-b_L-c₁-FeS-Q-Q the reaction (5) could be written as follows:(5a)here x designate any redox state of a respective transporter, 0 or 1. The expressions for reaction rates according to the mass action law are:(6)The program passes through the array for species (2), checks if the position of variable satisfy the requirements for substrate given in (5a) (that three last digits are 011), calculates the position of respective product in the array (three last digits are 101 and the digits designated as x are the same as for chosen substrate), then calculates fluxes (6) and adds them to the derivatives for the substrate and product. For the derivatives the program also has an array, where positions are organized in the same way as for concentrations.

For the other species capable to perform this reaction, Qi-Qi-b_H-b_L-c₁-FeS-Qo-Qo, it could be written asand reaction rates expressed similar to (6).

The model takes into account the ratio of forward and reverse rate constants, which could be defined from the difference of midpoint redox potentials (E_m):(6a)where n is the number of electrons transported, F = 96500 c/mol is the Faraday constant, R = 8.3 J/(mol×K) is the gas constant, T = 298 K is temperature. The derivation of this equation is based on the basic thermodynamic principles as is described in details in Text S1. The reported values for E_m at pH 7 vary as much as 312 mV, [25] or 280 mV, [26] for Fe³⁺/Fe²⁺ and 200 mV, [27] or 300 mV, [28] for Q-/QH2; this variability is reflected in the variability of value expressed by (6a) from 0.6 to 18.

Reaction 1 transports the electron received by Fe²⁺ of Rieske protein from QH₂ further to c₁:All the species (1)–(4) with reduced FeS center of Rieske protein can perform this reaction:Reaction rates for core species:(7)The ratio of forward and reverse rate constants is defined by an equation similar to (6a):(7a)Midpoint potentials, for Fe³⁺/Fe²⁺ of Riske protein is 312 mV [25] and for c₁ it is 341 mV [29]. This difference (ΔE_m = 29 mV) results in three-time difference in forward and reverse rate constants.

Reaction 2 transports the second electron of Qo to b_L:Only the species with ocupied Qo site perform this reaction:Reaction rates for b_H-b_L-c₁-FeS-Qo-Qo:(8)The reported E_m (−60 mV for b_L [30] and −140 mV for Q/Q⁻ [28]) giving ΔE_m = 80 mV according to the equation similar to (7a) define the ratio of forward to reverse rate constant as 25.

Reaction 3 transports electron from b_L to b_H All species with reduced b_L and oxidized b_H perform this reaction:Reaction rates for core species:(9)Since in this reaction electron moves in the direction from positive outer side to negative matrix side of the membrane, the rate constants depend of electric transmembrane potential [31].where ΔΨ is transmembrane potential.

Here the ratio of forward and reverse k⁰ is defined by midpoint redox potential difference (similar to (7a)). The reported values are −58 mV for b_L [30] and 61 mV for b_H [30]. This difference (ΔE_m = 119 mV) defines the forward rate constant to be two orders of magnitude higher than the reverse rate constant.

Reaction 4 transports first electron from b_H to Q on the n-side:This reaction is performed by the species with ubiquinone bound at Qi site:Reaction rates:(10)For b_H we accepted E_m = 61 mV, reported in [30]; for Q/Q⁻, the reported E_m vary from 90 mV [30] to 45 mV [32]. To start, we accepted the value which is close to E_m for b_H, in this case the forward and reverse rate constants are equal.

Reaction 5 transports second electron from b_H to Q⁻ on the n-side:This reaction can be performed by species that contain semiquinone bound on Qi site:Reaction rates:(11)For b_H E_m = 61 mV [30]; for Q⁻/QH₂ data in literature vary from 16.5 mV [30] to 150 mV [32]. To start, for this transition we also accepted the value close to E_m for b_H, in this case the forward and reverse rate constants are equal.

Reaction 6. Ubiquinol binding to and dissociation from the complex III at the cytosolic side (Qo) of the inner mitochondrial membrane. This is a way of transition between species: core (1) and species with occupied Qo site (2), and also (3) and (4):(12)The expressions for forward and reverse reaction rates are:(12a)Simulating this and other binding/dissociation reactions the program works with two arrays, since substrate and product are belong to different species. This does not change much the principles of automated calculation described for the reaction 0. In the same way the program checks the requirements for substrate position (in this case all redox form satisfy it) calculates the position of product specific for each redox form of substrate, calculates the rates (12a) and adds them to the respective derivatives.

For the reaction (12)As a starting point we assumed that C_xxxx = C_xxxx11 when QH₂ = Q. In this case K_d = 2 nmol/mg of protein.

Reaction 7. Ubiquinone binding and dissociation to the complex III at the matrix side of the inner mitochondrial membrane:(13)The expressions for reaction rates:(13a)In simulations we used the same value:Reaction 8. Ubiquinone dissociation and binding to the complex III at the cytosolic side of the inner mitochondrial membrane:The expressions for reaction rates:(14)Reaction 9. Ubiquinol dissociation and binding to the complex III at the matrix side of the inner mitochondrial membrane:The expressions for reaction rates:(15)Reaction 10. ROS production in the model.

Semiquinone bound to the Qo site of complex III could be oxidized by molecular oxygen, thus producing superoxide radical:This reaction is considered as unidirectional:(16)For relative ROS production k_ROS was taken to be 1 and K_ROS is specified in the legend to Figure 6.

Reaction 11. Succinate oxidation in complex II resulted in ubiquinone reduction (Q to QH₂), which rate V is described by the Michaelis' equation:(17)where K_Q = 0.5 nmol/mg of protein, K_S = 0.25 mM of protein, V_m = 1.88 nmol/mg of protein/s, and S is external succinate concentration. Thus, equation (17) represents succinate dehydrogenase activity in a generalized form not going into details as in above reactions.

Reaction 12. Electron transport from complex III to cytochrome c and further to complex IV.

All species (1)–(4) contribute to this rection:The expression for this reaction rate, which in fact integrates all the steps of electron transition from cytochrome c1 to oxygen, takes into account oxygen concentration:(18)here O₂ is a fraction of dissolved oxygen with respect to its content at equilibrium with atmospheric oxygen at normal pressure, k is reaction rate constant, K_m is Michaelis constant for interaction with oxygen. These parameters were adjusted so that the model describes limitation of electron flow by oxygen, which was found experimentally. According to the experimental data [22],[13], the electron flow is not limited by O₂ availability until the oxygen concentration falls to about 1 µM (<0.1% O2). The model reproduces these data if K_m = 0.03, and k = 25 s⁻¹. These parameters were used to calculate the oxygen dependence of semiquinone radical content presented in Figure 5A of the main text.

Complex III content is 0.4 nmol/mg of protein [33], and total ubiquinone content is 4 nmol/mg of protein [33].

The system of differential equations describes the evolution of concentrations (C) of all redox forms, which the program references as is described above. Derivative of each redox state is calculated as a sum of all the reaction rates of influx to or efflux from the considered state, which are calculated in accordance with the equations (6–18). For each type of reaction there is a special function, which, once called for a specie, passes from redox state 0 to the last one checking if the redox conditions permitting the reaction are satisfied and, if yes, calculates the flux and adds it (with respective sign) to the derivatives of concentrations of reagents. In this way, to simulate the operation of respiratory complex it is necessary just to check whether the functions simulating all the reactions performed by the species (1)–(4) are called, and these functions automatically would calculate the derivatives for all 400 redox states of complex III.

For the numerical solutions of differential equations the program implements 5th order Runge-Kutta, Bulirch-Stoer, implicit Bulirch-Stoer, Rosenberg methods [34]. The program and brief user guide is free available in http://www.bq.ub.es/bioqint/selivanov.htm

Similar technique is described in more detail elsewhere [35],[36].

Isolation of rat brain mitochondria - All procedures involving animals were approved by the Children's Hospital of Pittsburgh and were in compliance with “Principles of Laboratory Animal Care” and the current laws of the United States. Rat brain mitochondria were isolated from the cortex of adult Wistar rats. After removal, tissue was minced and homogenized in ice-cold isolation buffer I (IB I) which contained: 225 mM mannitol, 75 mM sucrose, 5 mM HEPES buffer (pH adjusted to 7.3 with KOH), 0.1 mg/ml fatty acid free BSA, 1 mM tetrapotassium EDTA and 12% Percoll. The homogenate thus obtained was carefully layered on the top of a discontinuous gradient of Percoll (24% and 42%) prepared using the same buffer. The preparation was then centrifuged at 31,000×g for 10 min. The fraction containing the mitochondria located between 42% and 24% Percoll was carefully withdrawn by a syringe and washed from Percoll twice by pelleting in IB I. The resulting mitochondrial suspension was diluted in isolation buffer II (IB II), which was same as IB I, except for the concentration of EDTA (0.1 mM) and lack of albumin, and spun down at 12,000×g for 10 min. The deposit of mitochondria was homogenized in IB II at a final protein concentration of ∼20 mg/ml and stored on ice until use. The protein concentration in the mitochondrial samples was determined using a Protein Assay kit (Pierce Chemical Company, Rockford IL) according to the manufacture's instructions. Mitochondria prepared in this way were active for at least 4–5 hours, as determined by their ability to maintain a stable transmembrane potential in the presence of oxidizable substrates.

Hydrogen peroxide measurements were performed in a stirred cuvette mounted in a Shimatzu RF-5301 spectrofluorimeter maintained at 37°C. Mitochondria (0.2 mg/ml of protein) were added to the incubation medium that contained: 125 mM KCl; 2 mM KH₂ PO₄; 2 mM MgCl₂; 10 mM Tris;10 mM HEPES (pH 7.0); 100 µM EGTA; 5 mM pyruvate and particular concentrations of succinate (0.05–5 mM) as the oxidizable substrates. Reporting fluorescent dye for H₂O₂ was Amplex red (2 µM) which increased fluorescence upon oxidation to resorufin in the presence of 1 U/ml of horseradish peroxidase (HRP) as previously described [36]. Measurements were carried out at excitation/emission wavelengths of 560 nm (slit 1.5 nm)/590 nm (slit 3nm), respectively. Amounts of H₂O₂ released by mitochondria were estimated by constructing calibration curves using known H₂O₂ concentrations in the standard incubation buffer together with Amplex red and HRP, but without mitochondria. Normoxia experiments were performed in a conventional square (10×10 mm), open cuvette. Anoxic experiments were performed in a cuvette with a narrow rectangular neck (10×3 mM). Anoxic conditions were made by bubbling the cuvette with N₂ and, once the reaction media was depleted of O₂, closing it with a Teflon lid. Resumption of oxygen supply was achieved by opening the cuvette and bubbling it with air.

Mitochondrial transmembrane potential, ΔΨ_m - was estimated using fluorescence quenching of the cationic dye safranine O. Since polarized mitochondria have a negative charge inside, positively charged molecules of safranine O are accumulated inside the matrix; increase in dye concentration inside the matrix leads to fluorescence quenching , thus a decrease in fluorescence corresponds to an increase of membrane potential. The excitation wavelength was 495 nm (slit 3nm) and emission 586 nm (slit 5nm), and the dye concentration used was 2.5 µM [36].

All procedures involving animals were approved by the Children's Hospital of Pittsburgh and were in compliance with “Principles of Laboratory Animal Care” and the current laws of the United States.