A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

1. Preparation of Gelatin-coated Coverslips

1. Place acid-washed glass coverslips (15 mm in diameter) in a sterile plastic 100 mm dish(es). Place 8-9 coverslips per dish and make sure they are not touching each other or the sides of the dish..

Note: Coverslips, needles and tweezers need to be sterile to eliminate possible contaminating microorganisms, as well as endotoxins that will affect cellular functions, including motility.

2. Weigh the gelatin powder and resuspend the powder in deionized water to make a gelatin solution with a final concentration of 0.5 g/300 ml. Next, the gelatin solution needs to be autoclaved.
3. Pipette 2-3 drops of gelatin (~100-150 ml) onto each coverslip.

Note: Be careful not to allow the gelatin to touch the dish or you will not be able to remove the coverslips from the dish.

4. Bake the gelatin-coated coverslips in the 100 mm dish in an oven at 90 °C for 10 min.
5. Remove excess gelatin by gentle pipetting.
6. Dry the coverslips in the 100 mm dish in the oven at 70 °C for 45 min.
7. Once dry, remove the coverslips from the 100 mm dish using a sterile, endotoxin-free needle and tweezers (the needle is used to gently nudge up the coverslip to make it accessible for the tweezers to gently remove).
8. Place individual gelatin-coated coverslips into separate wells of a 24-well dish.

2. Preparation of Colloidal Gold-coated Coverslips

1. Weigh an appropriate amount of chloroauric acid (tetrachloroauric acid trihydrate; HAuCl₄·3H₂O) and then resuspend in sterile deionized water to prepare a final 14.5 mM solution (toxic). Prepare 1.5 ml of the solution per 8-9 coverslips.

Warning: Chloroauric acid is harmful if swallowed, causes severe skin burns and eye damage and may cause an allergic skin reaction. Toxic if swallowed.

2. Weigh an appropriate amount of sodium citrate (trisodium dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate; Na₃C₆H₅O₇) and then resuspend the powder in deionized water to make a final 0.5% solution. Prepare 1 ml of the solution per 8-9 coverslips.

Warning: Sodium citrate may cause eye and skin irritation. It may also cause respiratory and digestive tract irritation.

3. In a sterile, endotoxin-free beaker combine 1.5 ml of the sterile 14.5 mM HAuCl₄ solution and 13.5 ml of sterile deionized water; the result should yield a faint yellow solution. The aforementioned volumes will allow for 8-9 colloidal gold coverslips to be made.
4. While continuously stirring, heat the solution on a hot plate until it begins to boil.

Warning: The heating of the solution should be performed in the fume hood, as vapors can be harmful/toxic. The vapors are destructive to the tissue of the mucous membranes and upper respiratory tract.

5. Remove the beaker from the hot plate.
6. While stirring, add 0.7 ml of the 0.5% sodium citrate solution. The aforementioned volume will allow for 8-9 colloidal gold coverslips to be made.
7. Keep stirring this combined solution for approximately 2 min (Note: The color of the solution will gradually change from faint yellow, to clear, to grey, to purple, to deep purple, before finally reaching a red wine or, preferably, rust color). This product is your colloidal gold solution.
8. Cool the colloidal gold solution in a 10 ml pipette for 1-2 min (in order to keep the gelatin layer on the coverslip from melting when the colloidal gold solution is added) and then add 1.0-2.0 ml of the colloidal gold solution onto each gelatin-coated coverslip previously placed in a 24-well dish(es) (the amount of the colloidal gold solution that you add to the gelatin-coated coverslips depends on how well the gold particles precipitated in the solution – see comments below).

Note: Because there might be variations in the efficiency of gold particle precipitation, it is advised to first add 0.5-1 ml of the colloidal gold solution to the gelatin-coated coverslips and then place the 24-well dish in the incubator for 0.5 hr. After this incubation time, the coverslips should be checked under the light microscope for the appropriate density of the gold particles on the coverslips. See Figure 1 for 20x microscopy images of examples of too low and too high a concentration of gold particles. See Figure 2 for 40x microscopy images of an ideal concentration of gold particles (at least for use with monocytes). The optimal density of the gold nanoparticles on the slide should be experimentally determined for each cell type used, as the characteristics of the different cells will dictate their strength of movement on the coverslips. If the concentration of gold particles is insufficient, add an additional 0.5-1 ml of the colloidal gold solution to the gelatin-coated coverslips.

Depending on the cell size, the appropriate concentration of gold particles can vary and, thus will ultimately depend on the size of the cell examined for motility. For example, with human monocytes being small-sized cells (~10-20 μm²⁸), a higher concentration of gold particles was required in our analyses. The appropriate distribution of gold nanoparticles provides the researcher with the ability to easily and efficiently distinguish the edges of cellular tracks. A concentration of gold particles that is too high hampers the ability of cells to move and therefore to accurately measure their motility, while a concentration of gold particles that is too low limits ones ability to delineate an accurate track of motility.

Important Note: If the synthesis of gold nanoparticles is problematic, synthesized gold nanoparticles are commercially available in sizes from 5 nm to 400 nm. Additionally, fluorescent microspheres have also been used in studies of cell motility²⁹. However, the use of these microspheres requires a fluorescent microscope for the analysis of cell migration.

9. Place the 24-well dish with colloidal gold-covered coverslips in an incubator at 37 °C and incubate from 1 hr to overnight.
10. After incubation, rinse/remove unbound gold by dipping the coverslips (3x) into sterile phosphate buffered saline (PBS; pH 7.4).
11. Place these colloidal gold-coated coverslips in a clean 12-well dish(es) containing PBS.
12. Store these coverslips at 4 °C until ready to use (parafilm the plate before placing in a refrigerator).

Important Note: Always keep the colloidal gold-coated coverslips in some type of liquid/media as the gold particles can flake off from the coverslips if allowed to dry. The colloidal gold coverslips should be used within 2-3 months from the date that they were made.

Quality Control: In a single phagokinetic track motility assay, it is critical to use coverslips that are characterized by a similar concentration of gold particles. This simple point will allow for the quantitative differences in cellular motility between samples to be solely due to the nature of the treatment and not the physical properties of the colloidal gold-coated coverslips. We favor using only coverslips made at the same time in individual experiments, although we have shown that as long as the density of the gold nanoparticles are equal, the use of coverslips from different preparations is appropriate.

3. Analysis of Cellular Motility

1. Place the colloidal gold-coated coverslips in an appropriate cellular media for the cells of interest.
2. Transfer appropriately treated cells onto the colloidal gold-coated coverslips placed in a 24-well dish(es)

Note: The number of cells transferred to the single well needs to be determined for each cell type studied. Ideally, cells need to be equally distributed on the colloidal gold-coated coverslip, which in turn will allow for the statistical analysis of only tracks created by a single cell. If cells are plated at too high a concentration, it becomes highly probable that overlapping tracks of multiple cells will be seen. Overlapping tracks cannot be accurately quantitated and, thus, they cannot be taken into account in the final analyses of the movement of the tested cells. Overlapping tracks as a result of the involvement of multiple cells are usually easily observed; as merged or crossed tracks (in the field of analysis) in which two or more cells can be observed in the same contiguous cleared area.

3. Incubate at 37 °C/5% CO₂ for 24 hr (or for any suitable time; e.g. we have analyzed monocyte motility between 6 hr and 24 hr post-treatment and endothelial cells at 12 hr post-treatment). The optimal time frame for determining and measuring cellular motility will vary with the cell type studied and, thus should be experimentally determined for each cell type (a good starting point, however, is 6 – 12 hr post treatment).

Note: If experimental colloidal gold-coated coverslips need to be stored and/or analyzed at a later time, the cells and gold nanoparticles need to be fixed on the coverslips. To accomplish this fixation step; following Step 3.3, first wash coverslips carefully 2 times with 1x PBS (dipping of coverslips is preferable, as a removal of gold nanoparticles from coverslips must be avoided), then use a standard cell fixation method, such as incubation with room temperature 3% paraformaldehyde. After a 15-min incubation, the 3% paraformaldehyde should be removed and the coverslips washed carefully 3 times with 1x PBS. The fixed coverslips can be stored in a refrigerator.

4. Using a light microscope, capture images of the tracks created by a single moving cell (Note: The magnification used to take pictures of cellular tracks will certainly vary depending on the cell type under investigation). Examples of cellular tracks created by non-motile and motile cells on colloidal gold-coated coverslips are shown in Figure 2.

Note: The gold nanoparticles of the size used in the phagokinetic track motility assay has been found to be completely nontoxic for cells³⁰. If necessary, the viability of the examined cells can be assessed by staining with trypan blue or examined for other markers of cellular viability. One would have to take into account the need for fixation, type of fixation, etc., if this step needs to be undertaken.

5. Using the freely available software, such as ImageJ software (http://rsbweb.nih.gov/ij/ ) or NIH Image (http://rsb.info.nih.gov/nih-image/), both developed at the National Institutes of Health, or ImageTool (http://ddsdx.uthscsa.edu/dig/itdesc.html) developed at the University of Texas Health Science Center at San Antonio, the average area (in arbitrary units) of colloidal gold cleared by 10-20 or more cells (per sample) is determined for each experimental arm from the captured images. Statistics can then be performed on the collected results. For example, results can be plotted as means ± the standard errors of the means (SEM) with Student’s t tests performed, and a P value of <0.05 used as the measure of statistical significance between samples. Figures 3 and 4 show steps in the analysis of the area of colloidal gold cleared by the cell.