The phagokinetic track assay, primarily used to investigate the patterns and direction of migrating fibroblasts (1), has also been adapted for the quantitative analysis of cell motility (2, 3). The assay is based on the principle that migrating cells ingest, push to one side, or collect on their dorsal surface small particles in their path. In the original method, as described by Albrecht-Buehler (1), glass coverslips are coated with bovine serum albumin and surfaced with particles of colloidal gold. Cells are then seeded onto the gold monolayers and as they migrate, a clear, particle-free phagokinetic track is formed, leaving a permanent record of their movement. Quantification of cell migration may be achieved using computer-assisted image analysis. The assay has been used in studies of the movement of a wide spectrum of cell types, including fibroblasts (1), neutrophils (4), keratinocytes (5), and endothelial cells (6).
However, while the assay per se gives a reliable indication of cell movement, the preparation of the gold colloid monolayer is a limiting factor in experimental design, proving to be both unreliable and time-consuming. A minor modification which involves coating the coverslips in gelatin as opposed to albumin allows increased adherence of the gold but with little improvement in the quality of particle layer (7). Here, we primarily report on a method which greatly improves on the quality of the gold coating used in the assay, together with the application of these monolayers as used in motility studies.
Briefly, coverslips (22 Ο« 22 mm) were boiled in 5% detergent (7X; ICN Biomedicals, High Wycombe, UK) and then washed in alcohol to remove the grease. After being dried, they were immersed in a gelatin solution at 90Β°C for 10 min (Sigma, 300 Bloom; 0.5 g in 300 ml distilled H 2 O). The coverslips were then dried by being placed in a 70Β°C oven for 45 min.
Colloidal gold suspension was prepared by adding 11 ml distilled H 2 O and 6 ml Na 2 CO 3 (36.5 mM) to 1.8 ml AuHCl 4 (14.5 mM). The mixture was heated to 95Β°C with constant stirring, at which point 1.8 ml of freshly prepared 0.1% formaldehyde solution was added; the temperature was maintained at 95Β°C. A