Migration assays were performed using a sterilized culture plate insert with 8-μm diameter pores (Millicell, Cat. # PI8P01250; Millipore, Billerica, MA).
For Rf/6a cells, 200 μL of EC suspension were added to the upper well of each culture insert placed in a 12-well culture plate (Costar, Corning, NY). F12 medium (with 1% penicillin/streptomycin) containing one of the biochemical stimuli (0.5, 1.0, and 100 μg/mL EDPs, 1.0 μg/mL bioactive hexapeptides VGVAPG [BPs], Elastin Products Company) was added to the well of the 12-well culture plate (i.e., the bottom surface of the insert). HBSS (1%) was used in medium as a negative control, and 20% FBS in medium was used again as a positive control. For some experiments, inclusion of an irrelevant protein (BSA, 1.25 μg/mL) was performed to determine the specificity of elastin’s effects. The lower concentrations of the EDPs and the BPs were chosen to be similar to concentrations used previously by Robinet et al.,
10 and the concentration of EDPs found in the serum of exudative AMD patients by Sivaprasad et al.
19 The 100-fold EDP concentration range was chosen to determine whether the degree of cellular response was dependent on the peptide concentration. All conditions were performed in triplicate wells. Cells were incubated for 8 hours at 37°C, 95% humidity, 5% carbon dioxide. After incubation, membranes containing cells were fixed in one-half strength Karnovsky’s fix (1/2 K) for at least 1 hour.
27 The membranes were rinsed 2 × 4 minutes in 100 mM cacodylate buffer (pH 7.4) followed by a 30 minute treatment with 1% osmium tetroxide diluted in cacodylate buffer (100 mM). The membranes were then rinsed in double distilled water and then dehydrated. Final dehydration step was achieved with 2 × 10 minute incubations in hexamethyldisilizane (HMDS; Electron Microscopy Sciences, Hatfield, PA). Polycarbonate membranes were mounted on metal stubs with silver mounting medium (Ted Pella, Redding, CA), and then sputter coated with gold ions (K550; Emitech, Ltd., Kent, UK). Membranes were observed using a scanning electron microscope (SEM; S-3400N; Hitachi, Tokyo, Japan) at the University of Iowa Central Microscopy Facility. Ten digital images of the membrane’s lower surface were collected for each experimental sample at 150× magnification in a masked fashion, resulting in 30 images per condition due to triplicate wells. Cells were counted for each image using ImageJ software, taking care to count only cells that have established contact onto the membrane beyond the pore opening. In other words, cells that occupied pores but had not attached a portion of their surface membrane to the polymer surface were not included in the counts. Pairwise comparisons were performed using the Student’s
t-test.
Migration assays were repeated using fourth passage purified human choroidal ECs instead of Rf/6a cells. Elastin fragments were used only at 100 μg/mL for this experiment.