The overall goal of this high-content microscopy-based method is to simultaneously quantify mitochondrial morphofunction and cellular levels of reactive oxygen species so as to establish a robust redox fingerprint for cell lines exposed to different experimental perturbations. This method can help answer key questions in redox biology such as whether pathogenic conditions or compound treatments elicit oxidative stress, and to what extent mitochondrial form and function are affected. The main advantage of this technique is that all parameters can be measured within the same cell at the same time.

To start the procedure, seed eight to 10, 000 human dermal fibroblasts in 60 inner wells of a black 96 well plate with a thin continuous polystyrene or glass bottom. When using different conditions, treatments, or cell lines, distribute their seeding locations homogeneously on the plate to minimize the plate effects. Then, seed a further eight to 10, 000 cells in well B01 to be used for focus adjustment.

Subsequently, fill the empty outer wells with medium to minimize the gradients between the wells and the environment. Gently tap the plate three times before placing it back into the incubator to avoid the cells from growing in patches. Culture the cells for 24 hours, or until they reach 70%confluency.

Afterward, save the treatment information for the experiment into a spreadsheet called setup. To set up the microscope, first calibrate the XY stage using the software. Next, create an imaging protocol for a multi well plate using the acquisition software.

This protocol allows automatic acquisition of set positions and selected wells of a 96 well plate. Now, select the correct type of multi well plate from a list of available plates provided within the software. Alternatively, define the multi well plate format using the plate and well dimensions.

Then, align the well plate by defining two corners of the four outer corner wells. Following that, select the wells that need to be acquired, and define an acquisition protocol consisting of a sequential wavelength acquisition. Make sure the GFP channel is set first.

Then, define a well plate loop to acquire four regularly spaced no overlapping images positioned around the center of each selected well using the defined acquisition protocol. In this step, prepare the staining solution for 60 wells by adding CM-H2DCFDA and TMRM stock solution to HBSS HEPES buffer. Next, discard the culture medium from the cells by turning the plate upside down in a single fluid motion.

Gently wash the cells twice with HH buffer. Don't forget well A01 with the cells used for focus adjustment. Discard the HH buffer in between the washing steps.

Then, load the cells with CM-H2DCFDA and TMRM by adding 100 microliters of the staining solution to each well. Again, don't forget well A01. Incubate the cells for 25 minutes in the dark at room temperature.

After 25 minutes, wash the cells twice with 100 microliters of HH buffer and add 100 microliters of HH buffer to all 60 inner wells. To perform the actual measurement, install the plate on the microscope. Then, turn on the hardware based autofocus system and adjust the autofocus offset using well B01 and the TMRM channel until you have a sharp image.

Subsequently, run the imaging protocol. The resulting image data set will provide information on basal ROS levels, mitochondrial membrane potential, and mitochondrial morphology. Next, to measure the induced ROS levels, carefully remove the 96 well plate from the microscope.

Add 100 microliters of TBHP solution at 40 micromolar to each well and wait at least three minutes to allow complete reaction of the TBHP with the CM-H2DCFDA. During this time, add 100 microliters of the antibody working solution to well A01. Now, mount the plate back on the microscope and check the focus again using well B01.

Then, acquire the same positions as in the first imaging round using the same imaging protocol. The resulting image data set will provide information on the induced ROS levels. Next, acquire flat field images in well A01.

Make sure the signals are well within the dynamic range. By adjusting the acquisition settings. Afterward, export the acquired data sets in a single folder as individual TIFF files using standardized nomenclature.

That includes reference to the plate, pre or post TBHP treatment, well, field, and channel separated by underscores. Also, save the flat field images in the same folder as individual TIFF files using the standardized nomenclature. In this step, open the image processing software and install redox metrics macroset.

Then, open the setup interface to set the analysis settings by clicking on the S button. Select the image type, the number of channels, and the well that was used for acquiring the flat field images. After that, indicate which channel contains cells or mitochondria.

Check the background and enhance checkboxes to perform background subtraction and contrast limited adaptive histogram equalization respectively. Next, define a sigma for gaussian and blurring of the cells and laplacian enhancement of mitochondria. Select an automatic thresholding algorithm and fill in the size exclusion limits.

Then, test the segmentation settings on a few selected images of the acquired data sets by opening them and clicking the C or M buttons in the menu for cell or mitochondria segmentation respectively. Following that, from the batch analysis on the folder of interest by clicking on the hash button and selecting the folder with the images. Verify the settings and press okay.

After the batch analysis, visually verify the segmentation performance on a verification stack by clicking on the V button and selecting the folder with images. Go through the verification stack to confirm if segmentation was successful. Initial analysis of the extracted data is done automatically with a complimentary Shiny application.

This allows rapid interpretation of the results. To start, open the application in R Studio. Choose to run it in an external browser.

On the input page, select the directory where the result files are located. Make sure the setup file is also present in this directory. The result files and setup information are automatically imported, compiled, and visualized.

The experimental setup page shows the layout of the experiment. The next page displays ROS levels as well as several mitochondrial parameters for each plate separately. Select the plate that you want to visualize using the dropdown menu.

Data is shown in a multi well plate format, as well as in box plots with outliers labeled with well and image number. The results whole experiment page shows the normalized results from all plates combined, together with a basic statistical analysis. If a drop file is supplied through the input page, the specified data points will be excluded.

On the cluster analysis page, a sensitive redox profile is composed using a principal component and analysis on data from five parameters. Finally, the download data page allows saving of the processed and rearranged data for further use. Shown here are the results from an experiment in which human dermal fibroblasts were treated with the HIV protease inhibitor Saquinavir.

Using the described protocol, a significant increase was detected for both basal and induced ROS levels. As compared to the control cells, treated with DMSO. Saquinavir also significantly affected mitochondrial morphofunction.

The mitochondrial membrane potential measured as the average TMRM signal per mitochondrial pixel significantly increased. In addition, mitochondria acquired a highly fragmented pattern, quantitatively indicated by a higher circularity and smaller average size of individual mitochondria. When combining the aforementioned parameters using principle component analysis, both the control and Saquinavir conditions could be clearly separated from each other by their redox fingerprints.

While attempting this procedure, it's important to remember that the illumination itself causes oxidated stress. Therefore, exposure conditions should be minimized and kept constant throughout the experiment. Once mastered, up to 20 96 well plates can be stained and acquired in one day.

Following the second imaging round, the cells can be fixed and used for a downstream immunofluorescent staining. This increases the number of measured parameters on the exact same cells, and allows answering additional questions. After watching this video, you should be able to perform the combined measurements of ROS and mitochondrial morphofunction in adherent cell cultures using high-content microscopy.