The overall goal of the following experiments is to study disease associated mitochondrial phenotypes in human fibroblasts using live cell imaging technique. This is achieved by first obtaining a skin biopsy and preparing it for tissue culture. First, human fibroblasts will propagate from the skin sample within the first two weeks.
Next, the fibroblasts are detached and seeded into chambered cover glass wells. After 24 to 48 hours, live cell imaging dies are applied to evaluate mitochondrial function morphology or autophagy by live cell imaging technique. Ultimately, software based analysis of the images can reveal morphological and functional mitochondrial parameters in living human fibroblasts.
The major advantage of using primary fibroblasts from patients carrying disease associated mutations compared to already existing cell models like tumor cell lines, is that fibroblasts reflect important pathological features of the human disease without previous genetic manipulation based on endogeneous protein levels. These techniques can help to identify early signs of Parkinson's disease, such as alterations in mitochondrial function and dynamics, and to define new therapeutic strategies, Begin with a fresh tissue biopsy, which has been taken by a biopsy punch. Place the specimen into a first dish with PBS and proceed with three washing steps by transferring it into three following dishes filled with PBS.
Cut the biopsy specimen into small pieces. Place two or three pieces into a T 25 flask by transferring them with a pipette. Together with PBS, prepare between two and four flasks from a single biopsy.
Carefully replace the PBS with a small amount of culture media so that the specimens can attach to the bottom of the flask. Incubate the cells until the first fibroblasts grow out of the epidermal sample. If growth problems are encountered, add fibroblast growth factor to enhance the cell's growth.
Once co fluency reaches 50 to 70%the cells are ready for use. If no experiment is planned, the fibroblasts should be frozen. Therefore, replace the culture media with DNA's enzymes after washing the cells with PBS and incubate them.
Take up the detached cells in culture, media and centrifuge. The cell suspension. Resuspend the obtained cell pellet in FCS with DMSO and freeze it in an isopropanol chamber at minus 80 degrees Celsius.
Experiments with human fibroblasts should generally be performed on cell lines with less than 10 passages. Always compare lines of the same passage number and ideally use multiple different genetically homogenous cell lines to compare with healthy controls. Begin the experiment by washing the attached fibroblasts once with PBS and then incubate with proteolytic DNA's enzymes to release the cells.
Count your cells with a counting chamber and seed 50 to 70, 000 cells in each well of your uncoated four chamber cover glass. After 24 to 48 hours, the live cell imaging experiment can be performed. Ensure that the live cell imaging microscope is equipped with an incubator that controls temperature and carbon dioxide levels during image acquisition.
An atmosphere of 37 degrees Celsius and 5%carbon dioxide should be maintained. Image the cells at 63 x or greater magnification using either confocal, laser scanning microscopy, or fluorescence microscopy with the aome technique as shown here. The autofluorescence of fibroblasts with higher passage numbers typically hampers fax analysis.
Therefore, the preferred method is live cell imaging. Microscopy 24 to 48 hours after seeding the cells in the chambered cover glass, change the medium to one with an MMP dependent dye due to light sensitivity work. Under diminished light conditions.
Incubate the cells protected from light for 15 minutes. Then replace the staining solution with our PMI medium without phenol. Red, proceed without a washing step and immediately image the cells when imaging the fibroblasts.
Avoid photobleaching by choosing a single exposure time between 200 and 300 milliseconds for all images. The image acquisition under standardized settings is very important as one would be unable to make comparisons based on intensity if the settings are different between cell lines. When first imaging positively stained fibroblasts, a clear TMRE signal is visualized.
After imaging this field of view, inevitable photo bleaching occurs, keep the next visual field adequately far from the photo bleached section using publicly available image J software. Analyze the images offline. Begin by selecting your cell of interest using a selection tool.
Convert the image to an eight bit image via the image type menu. Reduce all non-specific noise by using the D speckle function under the image menu. Choose the lookup tables fire condition under the image menu.
Switch in the threshold function to the over under function and adjust the threshold until the mitochondrial structures are well-defined. Under the analyze menu, select the analyze particles option to get a mean gray value for each detectable mitochondrial particle. Save the given result list as a spreadsheet.
Next, create a surface plot. Use the plugin interactive 3D surface plot in the 3D plugin section. Adjust the plot using different display options such as by changing the original colors to spectrum LUT to give the intensity scale bar for the appropriate plot.
In a separate program, open the spreadsheet and calculate the average of the mean gray value of the mitochondrial structures to measure mitochondrial morphology in fibroblasts. 24 to 48 hours. After seating the cells in a chambered cover glass, replace the media with one containing MIT tracker green due to light sensitivity work.
Under diminished light conditions, incubate the cells protected from light for 15 minutes. Then gently wash the cells once with PBS and replace the medium with fresh RPMI lacking phenol red. Now immediately image the cells as described in the previous section.
When imaging the fibroblasts to evaluate mitochondrial morphology. Be aware of setting up convenient parameters for imaging the mitochondrial structures with good quality. Pay attention to setting the right focus and choose an appropriate exposure time.
Like previous image analysis, use the same software to define regions of interest. Convert the image type to eight bit and reduce noise by using the D speckle function. Next, under the process and filter menu, use the convolution filter to highlight mitochondrial structures.
Then as before, adjust the threshold so that the signal to noise ratio is appropriate. As it is a critical step to set the threshold manually, it's mandatory to work with the close look on the original image files and to ensure proper image processing. Under the analyze menu, select the analyze particles option to automate the identification of mitochondrial structures based on a chosen pixel size.
Several mitochondrial parameters can then be subsequently calculated such as area perimeter, minor, and major axes or circularity. These measurements can be set individually under the analyze menu, save the given result list as a spreadsheet and open the file. Analyses of mitochondrial morphology can be carried out by defining the form factor indicating the degree of branching of a mitochondrial network.
Likewise, the aspect ratio defining the length of the mitochondria can be calculated to measure the mitochondrial lysosomal colocalization 24 to 48 hours after seeding the fibroblasts, change that to one. That includes mito tracker and lyo tracker. Pay attention to work protected from light.
Proceed to incubate the cells for 15 minutes in the dark. Then replace the media with RPMI, lacking phenol red, but still containing the same concentration of lysosomal stain. Proceed immediately to confocal imaging or fluorescence microscopy using the aome technique as red signals indicate lysosomes and green signals.
Visualize mitochondrial structures. Positive mitochondrial lysosomal colocalization is reflected by yellow signals due to overlap of both individual stainings later during the offline analysis. After selecting a region of interest, eight bit imaging and the Desp speckling option, choose the plugin Jacob as the colocalization analysis tool.
This operation outputs values for each of the channels and calculates the Pearsons coefficient overlap coefficient and mans coefficient. These values are used to estimate the lysosome and mitochondria colocalization. In particular, the Pearsons coefficient measures the linear relationship between two variables.
Mitochondrial function in living human fibroblasts was evaluated via the described imaging techniques. In functional assays, the TMRE fluorescence signal correlates with the mitochondrial membrane potential. Under normal MMP conditions, the TMRE fluorescence is significantly higher than under pathological conditions characterized by a reduced MMP.
This fluorescence intensity was measured by calculating the average of the mean gray value of every mitochondrial structure. The use of the option interactive surface plot. In Image J provides an illustration of different peak intensities in cells dyed with MIT tracker, green fm mitochondrial structures were visualized independent of their respective MMP.
The morphology is analyzed on the basis of binary figures. Mitochondrial lysosomal colocalization was ascertained by dyed cells with MIT tracker green fm. In conjunction with Lyo tracker red DND 99 colocalization is highlighted by the white arrows.
Image analysis was also used to determine the Pearsons coefficients of these overlaps lapse. After watching this video, you should have a good understanding of how to use human skin as ex vivo model as an important tool to characterize main pathological features of mitochondrial dysfunction during neurodegeneration.
