Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

The overall goal of the following experiment is to examine the influence of toxic chemicals on endothelial cell migration using different migration assays. First, the use of a modified avoidant chamber assay to examine the chemo kinesis of exposed endothelial cells is demonstrated in the next experiment. A wound healing assay is combined with cell tracking to provide additional in-depth information about the dynamics of cell migration.

In the final assay, investigation of the mitochondrial membrane potential is demonstrated for identifying the underlying mechanisms of impaired endothelial cell migration. Ultimately, these migration assays can be used to evaluate the effects of highly toxic alkylating agents on the mitochondrial membrane potential and migration of endothelial cells. The methods can help to answer key questions in the field of wound healing disorders induced by highly toxic chemicals demonstrating the procedures will be ramat, voer and Stefan Miller, all technicians from my lab To Preco the cell culture flasks begin by adding a sufficient volume of freshly prepared gelatin to just cover the bottom of a sterile cell culture flask.

Place the flask in a cell culture incubator for at least 30 minutes. Then remove the remaining gelatin solution and transfer the embryonic stem cell derived early endothelial cells or EEC in enough media to culture the cells to 80%co fluency when the cells have reached sub confluence. Remove the medium, rinse the cells with PBS and incubate them in one milliliter of Accutane per 25 square centimeter for two to 10 minutes.

When the cells have detached, split them at a one to five ratio in fresh medium for endothelial cell migration analysis by Boyden chamber assay. Pre-code, the filter inserts in 500 microliters of freshly prepared 0.1%gelatin. After harvesting the EEC as just demonstrated, count the cells and add 500 microliters of cell culture medium to the lower compartments of the boyden chamber.

Next, add exactly one times 10 to the fourth EEC in 500 microliters of medium to each filter. Then after removing any bubbles, place the Boyden chamber into the incubator for exactly eight hours. At the end of the incubation, rinse the filters one time with PBS.

Then fix the cells with the addition of 0.5 milliliters of 4%para formaldehyde to both compartments after 20 minutes. Wash the filters at least three times with 0.1 molar PBS and excise the membranes with the scalpel. Use mounting medium supplemented with DPI to mount each membrane between two glass cover slips.

Taking care to note the orientation of the filters. Then use a fluorescent microscope at a 400 x magnification to count only the cells that have migrated toward the lower compartment. Taking care not to confuse the pores with the migrating cells.

To initiate the wound healing assay, manually lift the cells from an 80%confluent EEC culture dish by gently pushing a sterile 10 microliter pipette tip across the surface of the culture in a smooth straight line from one side of the dish to the other. Wash the plate twice in 0.1 molar PBS to remove the detached cells. Then add medium supplemented with the compound of interest to the culture container.

Then mount the culture dish under a microscope capable of live cell imaging under cell culture conditions, and acquire time-lapse images over 24 hours at 10 minute intervals, using at least a 10 24 by 10 24 resolution at the end of the imaging session. Use the appropriate software tool to measure the gap width at zero and 24 hours to perform a cell tracking assay. Import the image sequence into image J and enable the MT track J image J plugin.

Then using the add command, select 10 cells randomly in the field of view to manually track the movement of the cells over the 24 hour imaging period. Use the measure command to display the results. Then use the movie command to export the tracks to assess the mitochondrial membrane potential of the cells after treatment with the experimental compound of interest, replace the cell culture medium with two microliters of freshly prepared TMRM in one milliliter of fresh cell culture.

Medium, then without washing immediately, place the dish under the microscope and obtain images of the control and experimentally treated cells without changing the acquisition parameters To ensure comparability between the different groups, wound healing requires angiogenesis, which are dependent on the migration of endothelial cells as illustrated by the graph exposure of the EEC to the alkylating agent. Chlorambacil results in a significant decrease in cell migration as measured by the Boyden chamber assay. The addition of the reactive oxygen species or ROS scavenger alpha-linolenic acid directly after 24 hour Chlorambacil exposure significantly rescues the phenotype almost to control levels.

The Boyden chamber assay, however, does not provide information about the migration behavior of the cells in this representative wound and healing assay. The unexposed EEC were able to close the gap within 24 hours while the chlorambacil treated EEC were not. Moreover, cell tracking of the individual EEC revealed that the chlorambacil exposed cells exhibited little movement, which was restored after treatment with the ROS scavenger.

In fact, chlorambacil exposed EEC exhibit A breakdown of their mitochondrial membrane potential as illustrated by their lack of TMRM expression. Treatment of the EEC with the ROS scavenger, however prevented this mitochondrial damage maintaining their mitochondrial membrane potential. After watching this video, you should have a good understanding of how to analyze migration of chemically disturbed endothelial cells by using a modified point in chamber assay by using a scratch wound assay and how to assess their mitochondrial membrane potential.