Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Neuroscience
 

Summary

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Cite this Article

Copy Citation | Download Citations

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).

Abstract

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.

Protocol

1. Cell culture

  1. Cortical neurons are isolated and grown using previously described techniques and plated on culture dishes with a glass bottom (MatTek Corporation, Ashland, MA ) coated with poly-D-lysine and laminin1.

2. Preparing the stock solutions for the fluorescent probes TMRM and H2DCF-DA

  1. Prepare a 10-mM stock solution of TMRM by dissolving 5.0 mg of TMRM in 1 ml of anhydrous dimethylsulfoxide. Vortex it for 1 min. Then, make aliquots and store them at -20°C, protect from light, and use within one month.
  2. Next, prepare a 10-mM stock solution of H2DCF-DA by dissolving 4.87 mg of H2DCF-DA in 1 ml of anhydrous DMSO. Similarly, vortex it for 1 min. Then, make aliquots and store them at -20°C, protect from light, and use within one week.

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.

  1. To load the rat cortical neurons with TMRM, first, wash the cultured neurons 3 times with Tyrode's buffer (Text Overlay: TB: 145 mM NaCl, 5 mM KCl, 10 mM glucose, 1.5 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES; adjust pH to 7.4 with NaOH). Then, prepare 20 nM of TMRM by diluting the 10 mM TMRM stock 1/1000 times in TB and then add 2 μl of diluted TMRM per 1 ml of TB. Incubate the neurons with TMRM for 45 min in the dark at room temperature. After 45 min, mount the culture dish on the stage of the microscope and start imaging.
  2. To load the rat cortical neurons with H2DCF-DA, wash the cultured neurons 3 times with TB. Next, prepare 2 μM of H2DCF-DA by diluting the 10 mM H2DCF-DA stock 1/10 times in TB and then add 2 μl of diluted H2DCF-DA per 1 ml of TB. Then, incubate the neurons with H2DCF-DA for 45 min in dark at room temperature. After 45 min, wash the neurons 4 times with TB to remove excess fluorescent indicator before obtaining images.

4. Live imaging of neurons incubated with TMRM to determine ΔΨm

  1. To perform live imaging of neurons incubated with TMRM, confocal laser scanning microscopy (Text Overlay: LSM 510, Carl Zeiss Inc.), with the application of live time-series program, is used. Apply low-resolution and attenuated laser power (Text Overlay: low resolution: 256 x 256; laser power: 1%) to minimize the time needed to obtain images and to avoid photobleaching.
  2. . Next, adjust the focus of the mounted neurons loaded with TMRM using reflected light. Examine the TMRM fluorescence by illumination at 514 nm and detection at 570 nm. Set the detection gain of a camera just below the saturation level.
  3. Once all parameters which include resolution, laser power, detection gain of a camera, and time-lapse interval to obtain images are set; do not change these settings between experiments. Next, change the field. Start collecting images.
  4. To test changes in ΔΨm, stimuli such as 1 μM of FCCP or 2 μg/ml of oligomycin, can be applied, which will significantly depolarize or hyperpolarize the mitochondrial membrane potential, respectively. These changes will be reflected by a decrease in TMRM fluorescence intensity compared with the baseline fluorescence intensity in the case of FCCP, or an increase in TMRM fluorescence intensity in the case of oligomycin.

5. Live imaging of neurons incubated with H2DCF-DA to determine ROS

  1. To perform live imaging of neurons incubated with H2DCF-DA, first, mount the culture dish on the stage of a microscope. Adjust the focus of the cells using reflected light. Examine DCF fluorescence by excitation at 488 nm and emission at 515 nm.
  2. Next, adjust the laser power to 5-7%, detector gain, and resolution of 256 x 256. Do not change these settings between experiments. Then, set the frequency for obtaining live images using the time series program.
  3. Select a new field and start acquiring images. To detect changes in ROS levels, treat cells with 100-200 μM of H2O2. This will be reflected by an increase in DCF fluorescence intensity compared with baseline level.

6. Data analysis

  1. Use region of interest (Text Overlay: ROI) tool from the LSM program to select the areas. Then, measure the TMRM or ROS fluorescence intensities. Select ROIs from mitochondrial regions or ROIs from the entire cell body in imaged cells to measure the fluorescence intensities of TMRM or ROS, respectively.
  2. Calculate the average fluorescence intensities from all ROIs of each cell for TMRM or from whole cell bodies for all imaged cells for ROS for each time point. Select regions next to the cells to calculate the background fluorescence intensity. Take several measurements and calculate the average background intensity.
  3. Subtract the average background fluorescence intensity from average fluorescence intensities of ROIs in each cell for each time point using Microsoft Excel. After subtracting background intensity, normalize the TMRM or DCF fluorescence intensity to the baseline fluorescence using this formula (Text Overlay: ΔF = F-Fo/Fo x 100, where F= fluorescence intensity at any time point, Fo= baseline fluorescence). Then, use the Sigma Plot program to generate the plot showing the changes in fluorescence intensity over time.

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
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

Discussion

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.

Disclosures

No conflicts of interest declared.

Acknowledgments

This work was supported by the National Institutes of Health (K22NS050137 to J.C.B.).

Materials

Name Company Catalog Number Comments
Glass bottom culture dish MatTek Corp. P35G-1.5-14-C
NbActive4 BrainBits NbActive4
TMRM Invitrogen T668
H2DCF-DA Invitrogen C400
NaCl Sigma-Aldrich S6191
KCl Sigma-Aldrich P3911
CaCl2∙2H2O Sigma-Aldrich C3306
MgCl2∙6H2O Sigma-Aldrich M2670
D-Glucose Sigma-Aldrich G6152
HEPES Invitrogen 15630

DOWNLOAD MATERIALS LIST

References

  1. Hilgenberg, L. G., Smith, M. A. Preparation of dissociated mouse cortical neuron cultures. J. Vis. Exp. (2007).
  2. Scaduto, R. C. Jr, Grotyohann, L. W. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys. J. 76, 469-477 (1999).
  3. Ward, M. W. The amyloid precursor protein intracellular domain (AICD) disrupts actin dynamics and mitochondrial bioenergetics. J. Neurochem. 113, 275-284 (2010).
  4. Gunasekar, P. G., Kanthasamy, A. G., Borowitz, J. L., Isom, G. E. NMDA receptor activation produces concurrent generation of nitric oxide and reactive oxygen species: implication for cell death. J. Neurochem. 65, 2016-2021 (1995).

Comments

15 Comments

  1. I have ² queries:

    1. U ddid not mention washing off of the TMRM containing media before imaging. DŒs that mean that there is TMRM present in the imaging media?

    ². If DCFDA is washed off and imaging being done, upon addition of H²O², how can the DCF fluorescence increase?

    Reply
    Posted by: Anonymous
    February 25, 2012 - 3:02 AM
  2. 1. We did not wash the neurons after TMRM loading. TMRM makes equilibrium between compartments (mitochondria-cytosol- extracellular buffer) in response to negative membrane potentials and a very low concentration of the dye has to be present in the imaging buffer throughout the experiment.
    ². The principle of H²DCF-DA is different as it is cell-permeable probe and trapped into the cytosol once the acetate group is cleaved by intracellular esterases. The access of dye from extracellular medium has to be washed out. Intracellular oxidation of DCF-DA by reactive oxygen species (generated by H²O² addition) results an increased DCF fluorescence.

    Reply
    Posted by: Anonymous
    February 26, 2012 - 12:42 AM
  3. Thanks for the response. However regarding DCFDA, if the dye is washed off, and then H²O² added to increase the ROS inside cells, where is the dye to react with the newly formed ROS? How will the fluorescence increase if there are no more molecules of DCF available for reaction?

    Reply
    Posted by: Anonymous
    February 29, 2012 - 9:42 AM
  4. When we incubate cells with H²DCFDA for an optimal time period, most of the dye molecules are trapped in cytosol and excess of dye has to be washed from extracellular buffer. The cytosolic dye will have enough molecules to produce many fold increase of fluorescence upon oxidation. Due to basal ROS activity some of the dye molecule gets oxidized but not all until cells undergo some stress which causes ROS formation. It is well known that H²O² addition to the cells result intracellular ROS formation and even H²O² itself can diffuse through the cell membrane and oxidize the cytosolic dye to increase the fluorescence.

    Reply
    Posted by: Dinesh J.
    March 3, 2012 - 12:43 AM
  5. Changes in plasma membrane potential will also lead to a decrease in both the mitochondrial and cytosolic TMRM signals, (because there is a step-wise concentration gradient mitochondrial > cytosol > medium). How do you think is the best way distinguish between a loss of signal due to mitochondrial depolarization vs. loss of signal due to plasma membrane depolarization?

    Reply
    Posted by: Anonymous
    March 5, 2012 - 9:28 PM
  6. A previous lab member has used KCl in the media to keep the plasma membrane potential constant so that changes in TMRM fluorescence are reflective of changes in mitochondrial potential only rather than a combination of both.

    Reply
    Posted by: Anonymous
    March 7, 2012 - 1:57 AM
  7. In non- quenched method (1 to 50nM, should be optimized for a particular cell type) majority of the TMRM fluorescence arise from mitochondria and any change in TMRM fluorescence (either single mitochondrion or entire cell) can be directly correlated to polarized state of mitochondria. Alternately the ratio of TMRM fluorescence from mitochondrial and non-mitochondrial regions in cell can be normalized to ratio of TMRM fluorescence at baseline and after FCCP addition. A parallel control experiment to monitor the TMRM fluorescence under plasma membrane depolarization (such as in presence of 30mM KCl) also can be performed.

    Reply
    Posted by: Dinesh J.
    March 7, 2012 - 12:50 PM
  8. For the DCFDA experiments, is the use of verapamil advisable to inhibit the efflux of DCF from cells?

    Can both TMRM and DCFDA be used at the same time or will one affect the read-out of the other?

    Reply
    Posted by: Anonymous
    March 9, 2012 - 2:31 AM
  9. We never tried using verapamil in our experiments but there are reports using this drug in DCFDA experiments.
    Yes TMRM and DCFDA can be used at the same time to monitor ROS formation and mitochondrial membrane potential.

    Reply
    Posted by: Dinesh J.
    March 11, 2012 - 1:30 AM
  10. Hi, I am curious how stable the preparation is in order to measure membrane potential. We have a 4 h protocol applied to cells in culture and at the end of this treatment we'd like to assess the membrane potential. Would you recommend loading TMRM before or after the 4 h procedure? I am afraid the membrane potential might change during the 45 min incubation. Therefore, it seems better to load TMRM before perhaps.

    Reply
    Posted by: Bela S.
    July 16, 2012 - 11:37 PM
  11. TMRM incubation time varies from one cell type to another. However, once the equilibrium is reached, TMRM fluorescence remains quite stable for long time. You can try loading TMRM before treatment and keep a parallel control loading without any treatment to rule out the change in TMRM fluorescence due to artifacts.

    Reply
    Posted by: Dinesh J.
    July 17, 2012 - 11:54 PM
  12. Thanks. Another question is whether you can easily convert the fluoresce intensity ratio to absolute membrane voltage.

    Reply
    Posted by: Bela S.
    July 24, 2012 - 2:22 PM
  13. We can not convert the TMRM fluorescence into absolute membrane voltage. The difference in TMRM fluorescence intensity is always a relative change between the experimental groups.

    Reply
    Posted by: Dinesh J.
    July 24, 2012 - 11:13 PM
  14. But I still have some queries . In your article only described the determination of mitochondrial membrane potential and oxygen species in primary cortical neurons, my query is if I want to know the relationship of my target drug and its concentration to the result , how should I go on my study? Which step should I give my compound to the cell? How can I determine the appropriate concentration? I really appreciate you can give me some advice. I am looking forward for your reply. Wish you have a nice day. Many thinks~

    Reply
    Posted by: longjun z.
    February 25, 2013 - 9:40 PM
  15. First, you will have to optimize the dye as well as the drug concentration in the cells independently. I would suggest optimizing the dye loading conditions first, then do dose dependent changes in the fluorescence of either TMRM or DCF to get an optimal drug concentration to start with. Once you have the optimized conditions, you can measure the ROS and membrane potential in two ways. 1. Incubate the cells with the probe; image them before and after the addition of drug. ². You can incubate the cells with drug and then load the cells with probe to monitor the effect of your drug. If longer incubation of drug is required, second method is preferred. Both the methods are well documented in the literature.

    Reply
    Posted by: Dinesh J.
    February 28, 2013 - 1:23 PM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics