Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

This research focuses on mitochondria dysregulation in neurodegenerative diseases. We believe that this research can be used to understand potential causes for disease onset that could lead to therapeutics combating neurodegenerative diseases like Parkinson's disease and ALS. Techniques such as super-resolution microscopy, like STED and SIM, or expansion microscopy have improved the ability to accurately understand protein distribution within individual organelles and mitochondria distribution throughout the cell.

Results of this technique can be used as a starting point to study the effect of Parkinson's disease linked mutations on mitochondrial turnover, and begin to understand the importance of regulating reactive oxygen species levels and mitochondrial membrane potential to maintain neuronal health. Our laboratory aims to mechanistically characterize individual mitochondrial quality control pathways to understand the interplay between these pathways. By having insights into pathway dynamics, one can understand how mitochondrial maintained and how mitochondrial dysregulation contributes to neurodegenerative disease onset.

Begin by mixing 200 microliters of reduced serum media with 2 micrograms of any one plaid DNA separately in a sterile micro centrifuge tube 1. Repeat this for two other plasmids in separate tubes. Then in the new tube 2, mix 200 microliters of reduced serum media with 6 microliters of transfection reagent and mix the content by pipetting.

Incubate the tubes for five minutes at room temperature before mixing the content of tube 2 to tube 1. In separate tubes, repeat the mixing of the other two plasmids with a transfection reagent. Incubate the mixture for 20 minutes at room temperature.

Next drop wise, add the transfection complexes to the HeLa cell culture seeded imaging dishes, ensuring equal distribution across the entire dish. One hour before imaging open the carbon dioxide tank valve and turn on the environmental controller for the microscope. Using the up and down arrows on the touch pad, adjust the temperature to 37 degrees Celsius and the carbon dioxide to 5%press Set when complete.

To adjust the laser settings, turn on the white light laser by clicking the Acquire tab and selecting Open Laser Overview. In the dialogue box, toggle the white light laser to On.Enter laser power as 85%Click the Excitation Control button and select Maximum Power from the dropdown menu. Begin the tetramethylrhodamine, ethyl ester percolate, or TMRE, experiment by setting the excitation laser to 514 nanometers and the emission spectra window to 524 to 545 nanometers for YFP.

Next, for MitoTracker Deep Red, set the excitation laser to 641 and the emission spectra window to 650 to 750 nanometers. Similarly, for TMRE, set the excitation laser to 555 nanometers and the emission spectra window to 557 to 643 nanometers. Begin the image acquisition setting by selecting the Acquisition tab and adjusting the format to 1024 x 1024.

Adjust the speed to 600 in the dropdown menu. Then, click the Line Average button and, from the dropdown menu, select 3. Turn bidirectional scanning on and set the phase and zoom factor to 22.61 and 1.50 respectively.

Once done, select the cells based on the YFP fluorescent signal by clicking on the YFP setting 1 and pressing Fast Live. Then adjust the gain and intensity of YFP and then select cells based on the YFP fluorescent signal. To image the DMSO control plate in the TMRE experiment, adjust the gain and intensity of the TMRE signal setting 2 so that the mitochondrial network intensity is just below saturation.

Keep the gain and intensity for TMRE constant. Then adjust the gain and intensity of the MitoTracker setting 1 so that the mitochondrial network is visible but dim. Once the gain and intensity settings are complete, click Start to acquire an image.

Acquire images of 20 cells per experimental condition. To measure the fluorescence intensity of the transfected HeLa cells in ImageJ software, click File and select Open. In the dialogue box, select the imaging files for the experiment and click OK.Once the Bio Formats Import Options window appears, select Split Channels and click OK.In tetramethylrhodamine, ethyl ester percolate or TMRE experiments, YFP is the first channel.

MitoTracker is the second channel, and TMRE is the third channel. Adjust the brightness by selecting Image, then Adjust and then the Brightness. Next, load the saved region of interest, or ROI by clicking Analyze, then Tools, then ROI Manager.

In the ROI Manager click More, then Open from the list and select the saved ROI. Then, measure the fluorescence intensity of five random regions in a single cell by selecting the saved ROI from the ROI Manager, and moving the ROI to a random location within a cell. To measure the fluorescence intensity, press M on the keyboard and repeat this with four additional non-overlapping regions.

When a dialogue box with the area and mean gray values appears, copy and paste the values into a spreadsheet for analysis. The results for the TMRE and MitoSOX fluorescence intensities showed that the treatment with the known uncoupling agent CCCP decreased the TMRE fluorescence intensity compared to the control conditions. In addition, 20 micromolar CCCP treatment induced superoxide production and increased the MitoSOX fluorescence intensity.

In mild or five micromolar CCCP stress conditions, the expression of Parkin Wild-Type and Parkin T240R resulted in higher TMRE intensity compared to the empty YFP control vector. The MitoSOX intensity was lower in cells expressing Parkin Wild-Type and Parkin T240R compared to cells expressing the YFP control vector, suggesting that Parkin expression helps maintain the mitochondrial network health by preserving higher mitochondrial membrane potentials and low superoxide levels.