2.8. Mitochondrial membrane potential (ΔΨm) and mtROS

Cell Preparation and Staining

1. Start by incubating H9c2 cells in a suitable culture flask at 37 °C with 5% CO2 in a dark environment. Use growth media supplemented with appropriate fluorescence dyes.

2. For measuring mitochondrial membrane potential (ΔΨm), stain the cells by adding growth media supplemented with 50nM Tetramethylrhodamine, methyl ester (TMRM). Allow the cells to incubate with TMRM for 30 minutes.

3. To assess mitochondrial reactive oxygen species (mtROS), treat the cells with growth media containing 2μM MitoSOX and incubate for 10 minutes.

4. For cell counting and nucleus staining, add growth media supplemented with 50 nM Hoechst 33342 to the cells and incubate for 30 minutes.

Live Cell Imaging

Prepare the Olympus IX73 microscope with a UPLANFL10X objective lens.

1. Set the lamp intensity to 3%.

2. Open the Cellsense Dimension software and configure the imaging settings:

    a. Adjust the sensitivity to ISO100.

    b. Set the exposure time to 200ms.

    c. Choose a resolution of 1600x1200 pixels for the images.

3. Use the appropriate filters for capturing fluorescence:

    a. For TMRM and MitoSOX, utilize the mCherry filter.

    b. Capture the Hoechst-stained nucleus using the DAPI filter.

Quantification of Fluorescence Intensity and normalization

1. Transfer the captured images to a computer with ImageJ software installed (downloadablefrom tttps://imagej.net/Download).

2. Open ImageJ and follow the steps to quantify fluorescence intensity:

3. Adjust the red channels corresponding to TMRM or MitoSOX fluorescence to make the intensity of pixels on the background minimum. All images should be adjusted with the same

value.

4. Utilize the particle analysis function provided in ImageJ (refer to https://imagej.net/imaging) to count and determine the number of cells based on the Hoechst-stained nuclei.

5. Measure the integrated pixel density of the red channel fluorescence for each cell.

6. Normalize the fluorescence intensity data by dividing the integrated pixel density by the number of cells.