Fibronectin

Nunc Lab Tek II 2-well chamber slides (0.7 cm²) were coated in the following extracellular matrix (ECM) components: fibronectin, collagen IV, matrigel (phenol red-free, growth factor reduced), and collagen I. fibronectin, collagen IV, and matrigel (BD Biosciences) were diluted to concentrations of 100 μg mL⁻¹ in phosphate buffered saline (PBS, Invitrogen). Wells were coated with 100 μL cm⁻² diluted ECM proteins for 2 hours at 4°C. Wells were washed once with PBS before cell seeding. Rat tail collagen I (BD) was diluted to a concentration of 100 μg mL⁻¹ in 0.02 M acetic acid. Wells were coated for 1 hour at 37°C and washed three times with PBS. Uncoated glass was used as a control. Astrocytes were seeded on the coated dishes at a concentration of 5,000 cells cm⁻². Low magnification images of cells on different surface coatings are shown in Figure S1. For the co-culture experiments, HBMECs were seeded onto 2-well chamber slides at a concentration of 50,000 cells cm⁻². Cells were grown to confluence for 48 hours in M199 media. After confluence was reached, the medium was replaced with DMEM/f12 and astrocytes were seeded on top of the monolayer at a density of 5,000 cells cm⁻². In all experiments, astrocytes were fixed after 24 hours. All experiments on coated surfaces were performed in triplicate.

Cells were kept in suspension or replated on ECM (10 μg/ml, laminin or fibronectin, BD Biosciences)-precoated dishes or coverglasses in the absence (in the case of fibronectin) or presence of low serum (2%, in the case of laminin) for 1 h before being analyzed for cell-ECM adhesion signaling by standard western blotting or for the formation of focal adhesions by indirect immunofluorescence, as described previously [27 (link)]. Function blocking antibodies against human integrin α6 (Millipore, 20 μg/ml) were preincubated with cells by rocking at 37°C for 1 h prior to replating. Pharmacological inhibitors were added to the culture media for 24 h or to replating media at the reseeding time.