Department of Molecular Pharmacology and Experimental Therapeutics, Loyola University Chicago
Joshi, D. C., Bakowska, J. C. Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons. J. Vis. Exp. (51), e2704, doi:10.3791/2704 (2011).
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria.
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.
1. Cell culture
2. Preparing the stock solutions for the fluorescent probes TMRM and H2DCF-DA
3. Loading rat cortical neurons with TMRM and H2DCF-DA
TMRM is a potentiometric, cell-permeable fluorescent indicator that accumulates in the highly negatively charged interior of mitochondria. It is important to use the low concentrations (10-50 nM range) of TMRM to avoid auto-quenching of mitochondrial TMRM. Then, the fluorescence signal of TMRM can be directly co-related to ΔΨm across the inner mitochondrial membrane. A loss of ΔΨm causes TMRM to leak from mitochondria resulting in a loss of fluorescence intensity. H2DCF-DA is cell-permeable probe converted into DCF-DA by intracellular esterases, and its oxidation results in fluorescent DCF. The final concentration of H2DCF-DA ranges between 2-10 μM and it should be tested empirically in neurons derived from different brain regions since high loading concentrations might result in the saturation of DCF fluorescence even in the absence of H2O2. The presence of any endogenous or exogenous oxidant (e.g., nitric oxide, hydrogen peroxide) will increase DFC fluorescence intensity. Below, we provide a protocol for loading rat cortical neurons with TMRM and H2DCF-DA.
4. Live imaging of neurons incubated with TMRM to determine ΔΨm
5. Live imaging of neurons incubated with H2DCF-DA to determine ROS
6. Data analysis
7. Representative Results
Figure 1A shows a fluorescence image of rat cortical neurons incubated with TMRM. Addition of FCCP, a mitochondrial uncoupler, leads to mitochondrial depolarization and a loss of TMRM fluorescence intensity (Fig. 1B). The baseline TMRM fluorescence level remains stable before addition of FCCP (the first 350 sec; Fig. 1C). Quantitative analysis of TMRM fluorescence changes over time shows a significant decrease in TMRM fluorescence after addition of FCCP (Fig. 1C).
Figure 1D shows the fluorescence image of rat cortical neurons loaded with DCF. Addition of H2O2 results in increased DCF fluorescence intensity in cell bodies (Fig. 1E). The baseline DCF fluorescence level is unchanged (the first 120 sec) before application of H2O2. Time-lapse measurements of DCF fluorescence show its steady levels, which increase after H2O2 treatment (Fig. 1F).
Figure 1. Assessment of mitochondrial membrane potential and ROS levels in live rat cortical neurons. (A) Representative fluorescence image of cortical neurons loaded with TMRM. After scanning the baseline TMRM fluorescence, neurons were treated with the protonophore FCCP (1 μM). To the right is a pseudocolor intensity bar of TMRM fluorescence with bright yellow and black representing maximum and minimum intensity, respectively. The loss of TMRM fluorescence from the mitochondrial regions indicates the collapse of ΔΨm upon FCCP treatment (panel B). The quantitative representation of change in TMRM fluorescence intensity at different time points before and after FCCP treatment is shown in panel C. (D) Fluorescence image of rat cortical neurons loaded with H2DCF-DA. After determining the baseline DCF fluorescence, cells were treated with 200 μM H2O2, and the change in DCF fluorescence was assessed. An increase in the DCF fluorescence reflects the increase in ROS levels upon H2O2 treatment (E). Quantitative analysis of change in DCF fluorescence, before and after H2O2 treatment, is shown in panel F. Scale bar = 10 μm
Video.7.1 – labmedia 2704_Joshi.avi
Live cell imaging of TMRM in cortical neurons before and after FCCP addition using 40X objective. The pseudocolor intensity shows a maximum (bright yellow, before FCCP addition) and decreased (red color, after FCCP addition) TMRM fluorescence intensity after FCCP addition. Click here to view video
Video. 7.5 - labmedia 2704_Joshi.avi
Live cell imaging of DCF in cortical neurons before and after H2O2 addition using 40X objective. The baseline DCF fluorescence has light green color in cell bodies and H2O2 addition increases the DCF fluorescence intensity to bright green color. Click here to view video
We have presented a step-by-step procedure describing how to determine ΔΨm and ROS in rat cortical neurons using the fluorescent indicators TMRM and H2DCF-DA, respectively. For other cell types, it is important to empirically determine the final concentration and loading time for TMRM or H2DCF-DA. In general, TMRM concentrations range from 20-200 nM, and the cell incubation time with TMRM varies from 20 to 60 min. The final concentration of H2DCF-DA ranges from 2-10 μM, and incubation of cells in a loading solution containing this indicator varies from 30-45 min.
It is important to optimize the laser power and scan speed of taking the images to avoid both photo-toxicity to the cells and changes in the fluorescence intensity (for example flickering of TMRM fluorescence) in the absence of any stimulus. The optimized optical settings should result in a fluorescence signal that is not over or under saturated (threshold) in the absence of stimulus. The optimal conditions to collect the images from a selected field at a particular laser power and a scan speed are achieved when there are no changes in the fluorescence intensity of the probe in the absence of any stimulus for 10-15 min of live imaging.
Other fluorescence probes to determine ΔΨm include rhodamine 123 and tetra methyl rhodamine ethyl ester (TMRE). However, they were found to inhibit the respiratory processes in isolated mitochondria2. Importantly, TMRM has no effect on mitochondrial respiration at low concentrations2 and has low phototoxicity and photobleaching3 compared with other probes. H2DCF-DA is a good indicator for ROS as it is well retained in the cells and recognizes several oxidant species, such as peroxides, super oxides, and nitric oxide4.
No conflicts of interest declared.
This work was supported by the National Institutes of Health (K22NS050137 to J.C.B.).
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