Begin by mixing 200 microliters of reduced serum media with two micrograms of any one plasmid DNA separately in a sterile micro centrifuge tube one. Repeat this for two other plasmids in separate tubes. Then in the new tube two, mix 200 microliters of reduced serum media with six microliters of transfection reagent and mix the contents by pipetting.
Incubate the tubes for five minutes at room temperature before mixing the content of tube two to tube one. 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 tetraethyl rhodamine 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 by 1024. Adjust the speed to 600 in the dropdown menu. Then click the Line Average button and from the dropdown menu, select three.
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 one 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 two 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 one 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.
