Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

The following protocol describes techniques for the investigation of early endothelial cell migration. The proper cultivation of vascular endothelial cells requires pre-coating of cell culture flasks with gelatin to ensure proper proliferation and maintenance of an endothelial phenotype.

1. Pre-coating of Cell Culture Flasks

1. Dissolve gelatin in 0.1 M PBS to a final concentration of 0.1%.
2. Autoclave the solution with parameters for liquid autoclaving (for details see instructions of the specific autoclave).
3. Add sufficient volume of the autoclaved gelatin solution to a sterile cell culture flask (e.g., at least 5 ml for a T25 cell culture flask).
4. Transfer the flasks into an incubator (37° C, 5% CO₂ is not required but does not interfere) for at least 30 min.
5. Remove the remaining gelatin solution. Use the pre-coated flasks subsequently (see cell cultivation) or store under sterile conditions until use.

2. Cell Cultivation of Early Endothelial Cells

Note: Embryonic stem cell derived early endothelial cells (EEC) were obtained from differentiated murine embryoid bodies by magnetic-activated cell sorting of the PECAM-1 positive cell fraction as described earlier^10,11.

1. Culture EEC on gelatin-coated cell culture dishes in DMEM supplemented with 15% FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 200 µM L-glutamine, 100 µM ß-mercaptoethanol and 1% non-essential amino acids. Handle cells under sterile conditions. Cultivate the cells with 5% CO₂ at 37 °C and 95% humidity until sub-confluence (max. 80%).
2. At sub-confluence, split the cells at a 1:5 ratio. Note: Detachment of endothelial cells is a critical step.
   1. Harvest the cells with RT accutase. Remove the media, rinse with PBS and add 1 ml of accutase per 25 cm².
   2. Incubate the flask at RT for 2-10 min until the cells have detached. Disperse the cells and transfer them to the desired application. Note: A chemical neutralization of the accutase is not required as it takes place when the seeded cells are stored in the incubator at 37 °C. However, accutase activity can also be decreased by adding DMEM containing FCS.

3. Boyden Chamber

Note: Boyden chamber assays are performed by using light-opaque polyethylene terephthalate insert systems with 8 µm pore size.

1. Pre-coat the filter inserts (that fit inside cell culture wells thus creating a Boyden chamber) by adding 500 µl of 0.1% gelatin dissolved in 0.1 M PBS for at least 30 min.
2. If cells are to be exposed to toxic chemicals, expose according to the specific experimental design before cell harvesting. Note: Cells were exposed to 12.5 µg chlorambucil/ml DMEM for 24 hr. With regard to the specific experimental design, instructions may vary.
3. Harvest EEC and determine the cell number by using a counting cell chamber. Note: Automatic counting devices can be used, but should be used with caution: manual cell counting is more accurate and is highly recommended.
4. Add 500 µl cell culture medium into the lower chamber compartment of the Boyden Chamber.
5. Add exactly 10⁴ EEC in 500 µl cell culture medium per filter insert in the upper chamber compartment. Eliminate bubbles.
6. Incubate the filter inserts in the incubator for exactly 8 hr.
7. Rinse with PBS once and replace the medium with 0.5 ml 4% paraformaldehyde in both compartments for 25 min for cell fixation. Wash the filter extensively but at least 3 times with 0.1 M PBS.
8. Excise the membranes with a scalpel.
9. Mount the membrane between two glass cover slips with mounting medium containing DAPI for nuclear staining. Pay attention to the orientation. Ensure that only cells that have migrated towards the lower compartment side of the membrane are counted.
10. Count cells that have migrated towards the lower compartment side of the membrane with a fluorescence microscope at 400X magnification. Do not confuse membrane pores with migrated cells (Figure 1A, 1B). Investigate a reasonable number of biological replicates (at least 3 biological replicates per condition).

4. Wound Healing Assay

1. Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead.
   Note: If using phase-contrast microscopy, plastic based dishes can also be used.
2. Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) until 80% confluence. Important: do not cultivate the cells to complete confluence.
3. Scratch the monolayers with sterile 10 µl pipette tips. Push the tip gently without too much pressure onto the dish surface and move it in a straight line smoothly from one side to the other.Wash the cells twice with 0.1 M PBS to remove detached cells.
4. Add a sufficient volume of medium to the culture dish. If applicable, add compounds that should be investigated.
   Note: 1.5 ml medium containing 15 ng/ml alpha linolenic acid was added.
5. Mount the culture dish under a microscope capable of live cell imaging. Ensure 5% CO₂, 37 °C and a humidified atmosphere.
   Note: Humidification is especially important to avoid medium evaporation.
6. Acquire time-lapse images over 24 hr at 10 min intervals. Plan for large file sizes. Note: A resolution of 512 x 512 pixels is usually sufficient; however, we recommend using images of at least 1,024 x 1,024.
7. Measure the gap width at t = 0 hr and at t = 24 hr using the length tool of the software provided with the microscope or use open-source software (e.g., ImageJ). Note: In general specific image acquisition and analysis software is provided by the manufacturer. Therefore, for technical details regarding the use of the software, refer to the manual.

5. Cell Tracking

1. Depending on the available equipment, carefully choose the cell culture dishes or plates: When using DIC microscopy, avoid plastic surface based culture dishes or well plates but use glass based devices instead.
   Note: If using phase-contrast microscopy, plastic based dishes can also be used.
2. Seed 5 x 10⁴ EEC in supplemented DMEM in a suitable cell culture device (e.g., 24-multiwell plate) and cultivate the cells with 5% CO₂ at 37 °C and 95% humidity for 1-2 days.
3. When cells have grown up to a 80% confluence, remove the media and culture the cells with new media in the presence of the respective test substances (e.g., 12.5 µg chlorambucil / ml DMEM). Always include control cells (treated with the solvent, e.g., ethanol) at 37 °C for a certain period of time (24 hr, depending on the individual assay system).
4. Mount the culture dish under a microscope capable of live cell imaging (37 °C, 5% CO₂ and 95% humidity). Acquire time-lapse images over 24 hr at predefined intervals. Acquire images at 10 min intervals.
5. Perform manually tracking of EEC by the use of the ImageJ plugin MTrackJ. Choose 10 cells randomly from the field of view and track their movements by adding a data point per point in time using the “Add” command of MTrackJ.

Note: MTrackJ is available for free at [Meijering, Mtrackj." http://www.imagescience.org/meijering/software/mtrackj/ “] and ImageJ is available at [Rasband, Imagej." http://rsbweb.nih.gov/ij/ “]. A detailed manual about the MTrackJ plugin is available at “http://www.imagescience.org”.

6. Live Cell Imaging/Assessment of Mitochondrial Membrane Potential

1. Cultivate EEC in a suitable cell culture device (e.g., 4 cm glass bottom petri dish suitable for live cell imaging) up to a 80% confluence.
   Important: Do not cultivate the cells to complete confluence.
2. If applicable, expose the cells to chemicals. Note: Cells were exposed to 12.5 µg/ml chlorambucil for 24 hr. With regard to the specific experimental design, instructions may vary.
3. Prepare a 10 mM stock solution of tetramethylrhodamine (TMRM) in DMSO. Protect from light. Note: The stock solution can be stored at -20 °C.
4. Dilute the stock solution in cell culture medium to a working solution with a concentration of 10 µM (1:1,000 dilution). Protect from light and use as soon as possible. Note: The working solution can be kept at RT for some time (>1 hr), however, preparation of a fresh working solution is highly recommended.
5. Add 2 µl of the working solution to 1 ml fresh cell culture medium (loading solution).
6. Load the cells by replacing the cell culture medium with the loading solution. Incubate for 15 min at 37 °C, 5% CO₂ and humidified atmosphere (incubator). Caution: Almost all fluorescence indicators are exported by living cells over time; therefore avoid prolonged loading or delayed analysis.
7. Without washing, place the dish under a microscope suitable for live cell imaging. Important: fluorescence indicators are highly sensitive to light, therefore, avoid unnecessary light exposure.
8. Acquire images without changing the acquisition parameters to ensure comparability between different images.